Pharmaceutical formulation of odevixibat

ABSTRACT

The invention relates to a pharmaceutical formulation, e.g. a paediatric formulation, of odevixibat, which comprises a plurality of small particles. The formulation may be used in the treatment of liver diseases such as bile acid-dependent liver diseases, and particularly cholestatic liver diseases such as biliary atresia, progressive familial intrahepatic cholestasis (PFIC), Alagille syndrome (ALGS) and paediatric cholestatic pruritus. The invention also relates to a process for the preparation of the pharmaceutical formulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Swedish Application No. 1850761-6,filed Jun. 20, 2018, and to Swedish Application No. 1850762-4, filedJun. 20, 2018, the disclosures of which are incorporated by referenceherein in their entireties.

TECHNICAL FIELD

The invention relates to a pharmaceutical formulation, e.g. a paediatricformulation, of odevixibat, which comprises a plurality of smallparticles. The formulation may be used in the treatment of liverdiseases, such as bile acid-dependent liver diseases, and particularlycholestatic liver diseases such as biliary atresia, progressive familialintrahepatic cholestasis (PFIC), Alagille syndrome (ALGS) and paediatriccholestatic pruritus. The invention also relates to a process for thepreparation of the pharmaceutical formulation.

BACKGROUND

The compound1,1-dioxo-3,3-dibutyl-5-phenyl-7-methylthio-8-(N—{(R)-α-[N—((S)-1-carboxypropyl)carbamoyl]-4-hydroxybenzyl}carbamoylmethoxy)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine(odevixibat; also known as A4250) is disclosed in WO 03/022286. Thestructure of odevixibat is shown below.

As an inhibitor of the ileal bile acid transport (IBAT) mechanism,odevixibat inhibits the natural reabsorption of bile acids from theileum into the hepatic portal circulation. Bile acids that are notreabsorbed from the ileum are instead excreted into the faeces. Theoverall removal of bile acids from the enterohepatic circulation leadsto a decrease in the level of bile acids in serum and the liver.Odevixibat, or a pharmaceutically acceptable salt thereof, is thereforeuseful in the treatment of liver diseases that are associated withelevated bile acid levels, and particularly in the treatment of rarepaediatric cholestatic liver diseases.

Odevixibat exhibits high potency and should be administered in lowdoses, such as ranging from about 40 to about 120 μg/kg. Thiscorresponds to doses as low as 200 to 800 μg in the treatment ofpaediatric patients that weigh about 5 to 20 kg (e.g., infants andtoddlers). It is desirable that a formulation of odevixibat can beadministered to young patients in a dosage form having a small size. Itis further desirable that such a formulation has good palatability, isnot perceived as gritty, and is well-tolerated by infants and smallchildren.

Multiparticulates can be administered to infants from birth if they areadministered with a liquid. For children aged approximately 6 months andolder (i.e. after weaning), the multiparticulates can be administered intheir solid form either directly into the mouth or mixed with semi-solidfood. Particle size, shape, texture, hardness, taste and dose volume(i.e., the number of particles) have been reported to be important foracceptability of multiparticulates by infants and children (Kozarewicz,Int. J. Pharm. 2014, vol. 469, pp 245-248). Various literature reviewshave been conducted on the acceptability of different oral dosage formsin paediatric and older adult patients (see e.g. Liu, et al., Drugs2014, vol. 74, pp. 1871-1889; Drumond et al., Int. J. Pharm. 2017, vol.521, pp. 294-305; Mistry et al., J. Pharm. Pharmacol. 2017, vol. 69, pp.361-376; Walsh et al., Int. J. Pharm. 2017, vol. 536, pp. 547-562), butthe size and/or dose volume (amount) of multiparticulates investigatedhave not always been reported in these reviews.

Perception of grittiness may be influenced by a range of factorsincluding particle size, quantity and dosing vehicle (see Mishra et al.,Yakugaku Zasshi 2009, vol. 129, pp. 1537-1544; Lopez et al., Eur. J.Pharm. Sci. 2016, vol. 92, pp. 156-162) as well as the hardness andshape of the particles (Tyle, Acta Psychologica 1993, vol. 84, pp.111-118), with irregular particles being perceived as larger than round(spherical) particles of the same size (Engelen et al., J. Text. Studies2005, vol. 36, pp. 373-386). Grittiness perception studies have shownthat grittiness scores may increase with increasing size and dose of themultiparticulates, whereas grittiness scores may decrease withincreasing vehicle viscosity (Lopez et al., Eur. J. Pharm. Sci. 2016,vol. 92, pp. 156-162).

Capsules can be acceptable for children from approximately 6 years ofage. The swallowability of the capsules can depend upon the dosage formdimensions (i.e. the size) and the ability of the child. The size,shape, taste and after taste are important capsule attributes that caninfluence patient acceptability (Kozarewicz, Int. J. Pharm. 2014, vol.469, pp 245-248). In some embodiments, the size of the capsules is keptas small as possible, and the number of capsules required per dose iskept to a minimum, e.g. not more than 1-3 capsules.

In view of the above, there is a need for a formulation of odevixibatthat can be easily administered in small doses adapted to the patients'weight. In some embodiments, the formulation should be suitable fortreating very young patients, should be easy to swallow, and should notbe perceived as gritty.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the X-ray powder diffractogram of dried crystalmodification 1.

FIG. 2 shows the X-ray powder diffractogram of an overhydrated sample ofcrystal modification 1.

FIG. 3 shows the drying of crystal modification 1, with the X-ray powderdiffractogram of an overhydrated sample of crystal modification 1 at thebottom and of a dried sample at the top (20 range 5-13°).

FIG. 4 shows the drying of crystal modification 1, with the X-ray powderdiffractogram of an overhydrated sample of crystal modification 1 at thebottom and of a dry sample at the top (20 range 18-25°).

FIG. 5 shows the X-ray powder diffractogram of crystal modification 2A,as obtained from a mixture of ethanol and water (70:30% v/v).

FIG. 6 shows the DSC trace of a sample of odevixibat with about 50%crystalline fraction (after pre-heating and cooling).

DETAILED DESCRIPTION OF THE INVENTION

Provided herein is a multiparticulate formulation containing low dosesof odevixibat. In some embodiments, the formulation is a paediatricformulation. In some embodiments, the formulation enables weight-baseddosing and can be sprinkled onto food. The formulation can be designedto have a good palatability, with an optimal balance between particlesize and dose volume.

In a first aspect, the invention relates to a pharmaceutical formulationof odevixibat, comprising a plurality of particles, wherein eachparticle contains odevixibat, or a pharmaceutically acceptable saltthereof, in an amount of from about 0.1% w/w to about 5.0% w/w based onthe total weight of the particle.

Because of the low doses in which odevixibat is to be administered, andfurther because of the multiparticulate form of the application, eachparticle of the formulation contains only a very low amount of theactive ingredient. For example, the amount of odevixibat, or apharmaceutically acceptable salt thereof, in each particle can be fromabout 0.2% w/w to about 3.5% w/w, preferably from about 0.3% w/w toabout 3.0% w/w, more preferably from about 0.4% w/w to about 2.5% w/w,and most preferably from about 0.5% w/w to about 2.0% w/w based on thetotal weight of the particle. In one preferred embodiment, each particlecontains odevixibat, or a pharmaceutically acceptable salt thereof, inan amount of about 0.5% w/w based on the total weight of the particle.In another preferred embodiment, each particle contains odevixibat, or apharmaceutically acceptable salt thereof, in an amount of about 1.0% w/wbased on the total weight of the particle. In yet another preferredembodiment, each particle contains odevixibat, or a pharmaceuticallyacceptable salt thereof, in an amount of about 1.5% w/w based on thetotal weight of the particle.

As used herein, the term “particles” refers to small particles rangingin size from about 0.1 to about 1.5 mm. Such particles are preferablyessentially spherical, although elongated or oblong particles also mightbe used. The particles may e.g. be pellets, beads, microparticles,microspheres, granules or minitablets, and may optionally be coated withone or more coating layers surrounding every such pellet, bead,microparticle, microsphere, granule or minitablet.

In some embodiments, the particles of the formulation are small enough,that they can be sprinkled onto food and easily swallowed. In someembodiments, the particles can be swallowed without causing a perceptionof grittiness. In some embodiments, the particles do not give thepatient an urge to chew the particles. The particles are, therefore,preferably between about 0.1 and about 1.5 mm in size, more preferablybetween about 0.1 and about 1.0 mm, and more preferably between about0.1 and 0.8 mm, such as about 0.2 mm, about 0.3 mm, about 0.4 mm, about0.5 mm, about 0.6 mm, or about 0.7 mm. In a more preferred embodiment,the particles are between about 0.4 and about 0.8 mm, such as about 0.5mm, or such as about 0.6 mm, or such as about 0.7 mm. In a particularembodiment of the invention, the particles are about 0.7 mm.

In some embodiments, the invention relates to a formulation ofodevixibat, wherein each particle comprises a core and a coating layersurrounding the core. The core of each particle may be a pellet, agranule, a minitablet, a bead, a microparticle or a microsphere.

In some embodiments, the core of each particle comprises the activepharmaceutical ingredient (odevixibat), while the coating layer of eachparticle does not comprise the active pharmaceutical ingredient. In someembodiments, the core of each particle comprises from about 0.1% toabout 5% w/w of the active pharmaceutical ingredient, based on the totalweight of the particle, such as from about 0.1% to about 2% w/w, such asfrom about 0.1% to about 1% w/w, or such as from about 0.1% to about0.5% w/w of the active pharmaceutical ingredient, based on the totalweight of the particle.

In some embodiments, the coating layer of each particle comprises theactive pharmaceutical ingredient (odevixibat), while the core of eachparticle does not comprise the active pharmaceutical ingredient. In someembodiments, the coating layer of each particle comprises from about0.1% to about 5% w/w of the active pharmaceutical ingredient, based onthe total weight of the particle, such as from about 0.1% to about 2%w/w, such as from about 0.1% to about 1% w/w, or such as from about 0.1%to about 0.5% w/w of the active pharmaceutical ingredient, based on thetotal weight of the particle.

The cores may be orally dispersible and comprise soluble ingredientssuch as a sugar (e.g., sucrose) or a soluble polymer (e.g. hydroxypropylmethylcellulose) or may be non-orally dispersible and comprisenon-soluble ingredients such as a non-soluble polymer (e.g.,microcrystalline cellulose). In a preferred embodiment of the invention,the cores comprise microcrystalline cellulose. In a more preferredembodiment, the cores are microcrystalline cellulose spheres.

The coating layer can further comprise a film-forming polymer, such as acellulose-based polymer, a polysaccharide-based polymer, anN-vinylpyrrolidone-based polymer, an acrylate, an acrylamide, orcopolymers thereof. Examples of suitable film-forming polymers includepolyvinyl alcohol (PVA), polyvinyl acetate phthalate (PVAP),polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), methacrylic acidcopolymers, starch, hydroxypropyl starch, chitosan, shellac, methylcellulose, hydroxypropyl cellulose (HPC), low-substituted hydroxypropylcellulose, hydroxypropyl methylcellulose (HPMC; or hypromellose),hydroxypropyl methylcellulose acetate succinate (HPMCAS), hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP),cellulose acetate trimellitate (CAT), as well as combinations thereof,such as a mixture of methyl cellulose and hydroxypropyl methylcellulose(metolose). In a preferred embodiment, the coating layer comprises afilm-forming polymer selected from the group consisting of hydroxypropylmethylcellulose, polyvinyl alcohol (PVA), polyethylene glycol (PEG),starch, hydroxypropyl starch and hydroxypropyl cellulose (HPC). In amost preferred embodiment, the coating layer comprises hydroxypropylmethylcellulose as the film-forming polymer.

The coating layer may optionally comprise one or more additionalingredients, such as a plasticizer (e.g. polyethylene glycol, triacetinor triethyl citrate), an anti-tack agent (e.g. talc or magnesiumstearate) or a colouring agent (e.g. titanium dioxide, iron oxides,riboflavin or turmeric).

In some embodiments, the formulation comprises odevixibat in crystallineform. In some embodiments, the formulation comprises a crystallinehydrate of odevixibat. In some embodiments, the formulation comprisescrystal modification 1 of odevixibat. This stable crystal modificationcan be obtained from a slurry of odevixibat in a mixture of water and anorganic solvent such as ethanol. Under these conditions, a mixed solvatecontaining about two moles of water and about one to about three, suchas about two to about three, moles of ethanol per mole of odevixibat(e.g., a dihydrate-diethanolate or a dihydrate-triethanolate) isinitially formed. This mixed solvate is referred to herein as crystalmodification 2. When crystal modification 2 is dried, such as undervacuum (e.g., less than 5 mbar) or under a nitrogen flow, it loses itsorganic solvent molecules and becomes crystal modification 1. In someembodiments, the transformation of crystal modification 2 to crystalmodification 1 proceeds via a crystalline intermediate. It is believedthat this crystalline intermediate is a dehydrated form, which quicklytakes up water from the air. While not wishing to be bound by theory, itis believed that the solvent molecules can be removed withoutdissolution and recrystallization of the crystals.

Crystal modification 1 of odevixibat cannot only be obtained from amixture of water and ethanol, as described above, but also from a slurryof odevixibat in a mixture of water and an organic solvent selected fromthe group consisting of methanol, 2-propanol, acetone, acetonitrile,1,4-dioxane, DMF and DMSO. Upon drying of the different mixed solvatesobtained under these conditions (crystal modification 2), the samecrystalline hydrate of odevixibat is obtained, namely crystalmodification 1.

Crystal modification 1 contains void volumes that are capable ofcontaining up to about 2 moles of water associated with the crystal permole of odevixibat, depending on the relative humidity. This form istherefore formally a channel hydrate. At about 30% relative humidity,however, crystal modification 1 contains a substantially stoichiometricamount of about 1.5 moles of water per mole of organic compound and isthus a sesquihydrate. The substantially stoichiometric amount of wateris considered advantageous, as the water content of the crystals remainssubstantially constant even with humidity changes within the normalrelative humidity range of about 30% to about 70% RH. Indeed, at normalhumidities, such as between about 30 and about 70% RH, crystalmodification 1 exhibits relatively low hygroscopicity.

In one embodiment, the formulation comprises crystal modification 1 ofodevixibat having an X-ray powder diffraction (XRPD) pattern, obtainedwith CuKα1-radiation, with at least specific peaks at °2θ positions5.6±0.2, 6.7±0.2 and/or 12.1±0.2.

In a specific embodiment, the formulation comprises crystal modification1 having an XRPD pattern, obtained with CuKα1-radiation, with specificpeaks at °2θ positions 5.6±0.2, 6.7±0.2 and 12.1±0.2 and one or more ofthe characteristic peaks: 4.1±0.2, 4.6±0.2, 9.3±0.2, 9.4±0.2 and10.7±0.2.

In a more specific embodiment, the formulation comprises crystalmodification 1 having an XRPD pattern, obtained with CuKα1-radiation,with specific peaks at °2θ positions 4.6±0.2, 5.6±0.2, 6.7±0.2, 9.3±0.2,9.4±0.2 and 12.1±0.2.

In a more specific embodiment, the formulation comprises crystalmodification 1 having an XRPD pattern, obtained with CuKα1-radiation,with characteristic peaks at °2θ positions 4.1±0.2, 4.6±0.2, 5.6±0.2,6.7±0.2, 9.3±0.2, 9.4±0.2, 10.7±0.2 and 12.1±0.2, and one or more of8.1±0.2, 8.6±0.2, 13.4±0.2, 13.8±0.2, 13.9±0.2, 16.6±0.2, 17.3±0.2,17.7±0.2, 18.3±0.2, 18.9±0.2, 19.4±0.2, 19.7±0.2, 20.5±0.2, 20.8±0.2,21.6±0.2, 23.2±0.2, 24.3±0.2, 29.8±0.2 and 30.6±0.2.

In an even more specific embodiment, the formulation comprises crystalmodification 1 having an XRPD pattern, obtained with CuKα1-radiation,with characteristic peaks at °2θ positions 4.1±0.2, 4.6±0.2, 5.6±0.2,6.7±0.2, 8.1±0.2, 8.6±0.2, 9.3±0.2, 9.4±0.2, 10.7±0.2, 12.1±0.2,13.4±0.2, 13.8±0.2, 13.9±0.2, 16.6±0.2, 17.3±0.2, 17.7±0.2, 18.3±0.2,18.9±0.2, 19.4±0.2, 19.7±0.2, 20.5±0.2, 20.8±0.2, 21.6±0.2, 23.2±0.2,24.3±0.2, 29.8±0.2 and 30.6±0.2.

In another embodiment, the formulation comprises crystal modification 1having an XRPD pattern, obtained with CuKα1-radiation, substantially asshown in FIG. 1.

Whereas crystal modification 1 is a sesquihydrate containing about 3.5%(w/w) water at about 30% relative humidity (based on the total crystalweight), it has been observed that the crystal can take up an additional1.5% (w/w) water when the humidity is increased up to 95% RH. Thesorption and desorption of this additional water is fully reversible.The additional water may be adsorbed on the surface or may further fillthe channels of the structure. In some embodiments, the term“overhydrated” refers to crystal modification 1 containing from about1.5 to about 4 moles of water per mole of odevixibat, such as from about1.5 to about 3.5, or such as from about 1.5 to 3, or such as from about1.5 to about 2.5, or such as from about 1.5 to about 2 moles of waterper mole of odevixibat. In some embodiments, the term “overhydrated”refers to crystal modification 1 containing from about 2 to about 4moles of water per mole of odevixibat, such as from about 2 to about3.5, or such as from about 2 to about 3, or such as from about 2 to 2.5moles of water per mole of odevixibat.

It has been observed that the XRPD pattern of overhydrated crystalmodification 1 slightly changes when it is dried, e.g. at 50° C. invacuum. A small shift of peaks is most clearly seen in the 20 ranges5-13° and 18-25°, as shown in FIGS. 3 and 4, respectively. Exposing thedried modification to elevated relative humidity, such as up to 95% RH,makes the XRPD pattern of the overhydrated modification appear again.The peak shifts are a result of the unit cell volume changes, whichoccur as water molecules go in and out of the crystal structure.

Therefore, in another embodiment, the formulation comprises overhydratedcrystal modification 1 having an X-ray powder diffraction (XRPD)pattern, obtained with CuKα1-radiation, with at least specific peaks at°2θ positions 5.7±0.2, 6.7±0.2 and/or 12.0±0.2.

In a specific embodiment, the formulation comprises overhydrated crystalmodification 1 having an XRPD pattern, obtained with CuKα1-radiation,with specific peaks at °2θ positions 5.7±0.2, 6.7±0.2 and 12.0±0.2 andone or more of the characteristic peaks: 4.0±0.2, 9.4±0.2, 9.6±0.2 and10.8±0.2.

In a more specific embodiment, the formulation comprises overhydratedcrystal modification 1 having an XRPD pattern, obtained withCuKα1-radiation, with specific peaks at °2θ positions 4.0±0.2, 5.7±0.2,6.7±0.2, 9.4±0.2, 9.6±0.2, 10.8±0.2 and 12.1±0.2.

In a more specific embodiment, the formulation comprises overhydratedcrystal modification 1 having an XRPD pattern, obtained withCuKα1-radiation, with characteristic peaks at °2θ positions 4.0±0.2,5.7±0.2, 6.7±0.2, 9.4±0.2, 9.6±0.2, 10.8±0.2 and 12.1±0.2, and one ormore of 4.7±0.2, 8.0±0.2, 8.6±0.2, 13.3±0.2, 14.1±0.2, 15.3±0.2,16.5±0.2, 17.3±0.2, 19.3±0.2, 19.7±0.2, 19.9±0.2, 20.1±0.2, 20.8±0.2,21.7±0.2, 23.6±0.2, 26.2±0.2, 26.5±0.2, 28.3±0.2 and 30.9±0.2.

In an even more specific embodiment, the formulation comprisesoverhydrated crystal modification 1 having an XRPD pattern, obtainedwith CuKα1-radiation, with characteristic peaks at *2θ positions4.0±0.2, 4.7±0.2, 5.7±0.2, 6.7±0.2, 8.0±0.2, 8.6±0.2, 9.4±0.2, 9.6±0.2,10.8±0.2, 12.1±0.2, 13.3±0.2, 14.1±0.2, 15.3±0.2, 16.5±0.2, 17.3±0.2,19.3±0.2, 19.7±0.2, 19.9±0.2, 20.1±0.2, 20.8±0.2, 21.7±0.2, 23.6±0.2,26.2±0.2, 26.5±0.2, 28.3±0.2 and 30.9±0.2.

In another embodiment, the formulation comprises overhydrated crystalmodification 1 of odevixibat having an XRPD pattern, obtained withCuKα1-radiation, substantially as shown in FIG. 2.

It is desirable that the use of organic solvents in the preparation ofthe formulation is avoided. In some embodiments, water is used as thesolvent for the preparation of the formulation. Odevixibat dissolves inwater only very poorly, and the solubility at pH 7 and at 37° C. hasbeen determined to be as low as about 30 μg/mL. Because of this lowsolubility in water, aqueous suspensions of odevixibat can containlarger agglomerates of odevixibat, which may lead to an unevendistribution of the active pharmaceutical ingredient on the cores, i.e.the cores may contain different amounts of odevixibat, which in turnimpacts dose uniformity. Accordingly, in some embodiments, the aqueoussuspension of odevixibat is homogeneous. In some embodiments, ahomogeneous aqueous suspension of odevixibat is sprayed onto the cores.

Odevixibat exhibits high potency and it should be administered in lowdoses, especially in the treatment of pediatric patients that weighabout 5 to 20 kg. In order to reach high dose uniformity for themultiparticulate formulation disclosed herein, it is important that eachparticle of the formulation substantially contains the same amount ofodevixibat, i.e., the deviation in the odevixibat content of theparticles of the formulation should be as low as possible.

As used herein, the term “homogeneous” refers to a suspension that doesnot contain agglomerates of odevixibat that are larger than about 200μm, and preferably no agglomerates larger than about 100 μm, morepreferably no agglomerates larger than about 50 μm. The size of theodevixibat agglomerates in the coating suspension may be determined byoptical microscopy, using a method based on European Pharmacopoeia 9.0,monograph 2.9.37, and as described in the experimental section.Alternatively, the size of the odevixibat agglomerates in the coatingsuspension may be determined by light scattering techniques, such aslow-angle laser light scattering (LALLS). In some embodiments, the d₉₀value for the particle size distribution of the coating suspension issmaller than 15 μm, such as smaller than 14 μm, such as smaller than 13μm, such as smaller than 12 μm, such as smaller than 11 μm, or such assmaller than 10 μm.

In some embodiments, a homogeneous suspension of odevixibat can beprepared by dispersing the compound in water by wet-milling. Wet-millingis a process in which a solid substance is dispersed in a liquid byshearing, by crushing, or by attrition. Examples of wet-millingapparatus include colloid mills, conical mills, ball mills, disc millsand high-shear dispersing machines. A specific example of a wet-millingapparatus for use in the present invention is a colloid mill.

In some embodiments, the crystallinity of odevixibat increases duringthe wet-milling.

Preferably, odevixibat is first wetted in a small amount of water usinga homogenizer and thereafter dispersed in water using a colloid mill.Spraying the homogenized dispersion onto the cores enables an evendistribution of the active pharmaceutical ingredient.

It is desirable that the formulation is free of any ingredients that arenot strictly necessary for the formulation, such as surfactants. In apreferred embodiment, therefore, the coating suspension does not containsurfactants. Similarly, in some embodiments, the coating layer of theformulation does not contain surfactants.

In one embodiment, the particles are contained within a sachet. Inanother embodiment, the particles are contained within a capsule. Suchcapsules may be made from gelatine, from a cellulose-based polymer suchas a hydroxypropyl methylcellulose (hypromellose), or from apolysaccharide-based polymer such as a pullulan. Capsules may beswallowed intact, or may be designed to be opened, so that, for example,the contents (i.e. the particles) can be sprinkled onto a food vehiclefor administration. In the latter case, the number of particles in onecapsule should preferably fit onto a single tablespoon of food. In someembodiments, a capsule contains from about 20 to about 100 mg ofparticles, such as about 30, about 40, about 50, about 60, about 70,about 80 or about 90 mg.

For younger paediatric patients, such as infants, toddlers and childrenup to about 6 years old, the particles are preferably sprinkled ontofood that can be easily swallowed and which does not require chewing,such as yoghurt, apple sauce, fruit purée or oatmeal. For olderpaediatric patients, such as children older than about 6 years old,adolescents and younger adults, capsules containing the particles may beswallowed intact, i.e. without opening. For newborn patients up to about6 months old, who have not yet been weaned or are unable to takesemi-solid food, the formulation can be administered by dispersing theparticles in a suitable liquid vehicle, such as breast milk, babyformula or water. When the particles have been dispersed in a liquidvehicle, they can be administered to the patient within 30 minutes afterdispersion, without loss of the active ingredient or indications ofdegradation. In some embodiments, the volume of liquid vehicle used foradministering the odevixibat particles, including rinsing, can besmaller than about 20 mL, such as smaller than about 15 mL, such assmaller than about 10 mL, or such as smaller than about 5 mL. In someembodiments, the dispersed particles are administered directly into themouth using an oral syringe.

The formulation disclosed herein may be used in the treatment orprevention of liver diseases, such as bile acid-dependent liverdiseases. In some embodiments, a liver disease involves elevated levelsof bile acids in the serum and/or in the liver. The formulationdisclosed herein may in particular be used in the treatment orprevention of cholestatic liver diseases, including rare paediatriccholestatic liver diseases, such as biliary atresia; post-Kasai biliaryatresia; post-liver transplantation biliary atresia; progressivefamilial intrahepatic cholestasis (PFIC), including PFIC-1, PFIC-2,PFIC-3 and non-specified PFIC, post-biliary diversion PFIC andpost-liver transplant PFIC; Alagille syndrome (ALGS); and primarybiliary cirrhosis (PBC); as well as paediatric cholestatic pruritus. Inone aspect, therefore, the invention relates to the formulationdisclosed herein for use in the treatment or prevention of a cholestaticliver disease. In another aspect, the invention relates to a method oftreating or preventing a cholestatic liver disease in a subject, such asa human, comprising administering to the subject in need of suchtreatment or prevention a therapeutically effective amount of theformulation disclosed herein.

Biliary atresia is a rare pediatric liver disease that involves apartial or total blockage (or even absence) of large bile ducts. Thisblockage or absence causes cholestasis that leads to the accumulation ofbile acids that damages the liver. In some embodiments, the accumulationof bile acids occurs in the extrahepatic biliary tree. In someembodiments, the accumulation of bile acids occurs in the intrahepaticbiliary tree. The current standard of care is the Kasai procedure, whichis a surgery that removes the blocked bile ducts and directly connects aportion of the small intestine to the liver. There are currently noapproved drug therapies for this disorder.

Provided herein are methods for treating biliary atresia in a subject inneed thereof, the methods comprising administration of a therapeuticallyeffective amount of the formulation disclosed herein. In someembodiments, the subject has undergone the Kasai procedure prior toadministration of the formulation disclosed herein. In some embodiments,the subject is administered the formulation disclosed herein prior toundergoing the Kasai procedure. In some embodiments, the treatment ofbiliary atresia decreases the level of serum bile acids in the subject.In some embodiments, the level of serum bile acids is determined by, forexample, an ELISA enzymatic assay or the assays for the measurement oftotal bile acids as described in Danese et al., PLoS One. 2017, vol.12(6): e0179200, which is incorporated by reference herein in itsentirety. In some embodiments, the level of serum bile acids candecrease by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to70%, 50% to 80%, or by more than 90% of the level of serum bile acidsprior to administration of the formulation disclosed herein. In someembodiments, the treatment of biliary atresia includes treatment ofpruritus.

PFIC is a rare genetic disorder that is estimated to affect between onein every 50,000 to 100,000 children born worldwide and causesprogressive, life-threatening liver disease.

One manifestation of PFIC is pruritus, which often results in a severelydiminished quality of life. In some cases, PFIC leads to cirrhosis andliver failure. Current therapies include Partial External BiliaryDiversion (PEBD) and liver transplantation, however, these options cancarry substantial risk of post-surgical complications, as well aspsychological and social issues.

Three alternative gene defects have been identified that correlate tothree separate PFIC subtypes known as types 1, 2 and 3.

-   -   PFIC, type 1, which is sometimes referred to as “Byler disease,”        is caused by impaired bile secretion due to mutations in the        ATP8B1 gene, which codes for a protein that helps to maintain an        appropriate balance of fats known as phospholipids in cell        membranes in the bile ducts. An imbalance in these phospholipids        is associated with cholestasis and elevated bile acids in the        liver. Subjects affected by PFIC, type 1 usually develop        cholestasis in the first months of life and, in the absence of        surgical treatment, progress to cirrhosis and end-stage liver        disease before the end of the first decade of life.    -   PFIC, type 2, which is sometimes referred to as “Byler        syndrome,” is caused by impaired bile salt secretion due to        mutations in the ABCB11 gene, which codes for a protein, known        as the bile salt export pump, that moves bile acids out of the        liver. Subjects with PFIC, type 2 often develop liver failure        within the first few years of life and are at increased risk of        developing a type of liver cancer known as hepatocellular        carcinoma.    -   PFIC, type 3, which typically presents in the first years of        childhood with progressive cholestasis, is caused by mutations        in the ABCB4 gene, which codes for a transporter that moves        phospholipids across cell membranes.

In addition, TJP2 gene, NR1H4 gene or Myo5b gene mutations have beenproposed to be causes of PFIC. In addition, some subjects with PFIC donot have a mutation in any of the ATP8B1, ABCB11, ABCB4, TJP2, NR1H4 orMyo5b genes. In these cases, the cause of the condition is unknown.

Exemplary mutations of the ATP8B1 gene or the resulting protein arelisted in Tables 1 and 2, with numbering based on the human wild typeATP8B1 protein (e.g., SEQ ID NO: 1) or gene (e.g., SEQ ID NO: 2).Exemplary mutations of the ABCB11 gene or the resulting protein arelisted in Tables 4 and 5, with numbering based on the human wild typeABCB11 protein (e.g., SEQ ID NO: 3) or gene (e.g., SEQ ID NO: 4).

As can be appreciated by those skilled in the art, an amino acidposition in a reference protein sequence that corresponds to a specificamino acid position in SEQ ID NO: 1 or 3 can be determined by aligningthe reference protein sequence with SEQ ID NO: 1 or 3 (e.g., using asoftware program, such as ClustalW2). Changes to these residues(referred to herein as “mutations”) may include single or multiple aminoacid substitutions, insertions within or flanking the sequences, anddeletions within or flanking the sequences. As can be appreciated bythose skilled in the art, an nucleotide position in a reference genesequence that corresponds to a specific nucleotide position in SEQ IDNO: 2 or 4 can be determined by aligning the reference gene sequencewith SEQ ID NO: 2 or 4 (e.g., using a software program, such asClustalW2). Changes to these residues (referred to herein as“mutations”) may include single or multiple nucleotide substitutions,insertions within or flanking the sequences, and deletions within orflanking the sequences. See also Kooistra, et al., “KLIFS: A structuralkinase-ligand interaction database,” Nucleic Acids Res. 2016, vol. 44,no. D1, pp. D365-D371, which is incorporated by reference in itsentirety herein.

TABLE 1 Exemplary ATP8B1 Mutations Amino acid position 3 (e.g., T3K)²⁷Amino acid position 23 (e.g., P23L)⁵ Amino acid position 45 (e.g.,N45T)^(5,8,9) Amino acid position 46 (e.g., R46X)^(A,25) Amino acidposition 62 (e.g., C62R)²⁸ Amino acid position 63 (e.g., T63T)⁴¹ Aminoacid position 70 (e.g., D70N)^(1,6) Amino acid position 71 (e.g.,R71H)⁴³ Amino acid position 78 (e.g., H78Q)¹⁹ Amino acid position 82(e.g., T82T)⁴¹ Amino acid position 92 (e.g., Y92Y)⁴¹ Amino acid position93 (e.g., A93A)⁶ Amino acid position 96 (e.g., A96G)²⁷ Amino acidposition 114 (e.g., E114Q)⁸ Amino acid position 127 (e.g., L127P⁶,L127V³⁶) Amino acid position 177 (e.g., T177T)⁶ Amino acid position 179(e.g., E179X)²⁹ Δ Amino acid positions 185-282⁴⁴ Amino acid position 197(e.g., G197Lfs*10)²² Amino acid position 201 (e.g., R201S²⁷, R201H³⁵)Amino acid position 203 (e.g., K203E^(5,8), K203R⁹, K203fs²⁵) Amino acidposition 205 (e.g., N205fs⁶, N205Kfs*2³⁵) Amino acid position 209 (e.g.,P209T)⁴ Amino acid position 217 (e.g., S217N)⁴³ Amino acid position 232(e.g., D232D)³⁰ Amino acid position 233 (e.g., G233R)³⁸ Amino acidposition 243 (e.g., L243fs*28)³³ Amino acid position 265 (e.g., C265R)²⁵Amino acid position 271 (e.g., R271X¹³, R271R³⁰) Amino acid position 288(e.g., L288S)⁶ Amino acid position 294 (e.g., L294S)⁴³ Amino acidposition 296 (e.g., R296C)¹¹ Amino acid position 305 (e.g., F305I)²⁸Amino acid position 306 (e.g., C306R)²³ Amino acid position 307 (e.g.,H307L)³⁵ Amino acid position 308 (e.g., G308V¹, G308D⁶, G308S³⁵) Aminoacid position 314 (e.g., G314S)¹³ Amino acid position 320 (e.g.,M320Vfs*13)¹¹ Amino acid position 337 (e.g., M337R)¹⁸ Amino acidposition 338 (e.g., N338K)¹⁸ Amino acid position 340 (e.g., M340V)¹⁸Amino acid position 344 (e.g., I344F)^(6,20) Amino acid position 349(e.g., I349T)⁴¹ Amino acid position 358 (e.g., G358R)²⁸ Amino acidposition 367 (e.g., G367G)⁴¹ Amino acid position 368 (e.g., N368D)⁴¹Amino acid position 393 (e.g., I393V)²⁷ Amino acid position 403 (e.g.,S403Y)⁶ Amino acid position 407 (e.g., S407N)⁴⁰ Amino acid position 412(e.g., R412P)⁶ Amino acid position 415 (e.g., Q415R)²⁷ Amino acidposition 422 (e.g., D422H)³⁵ Amino acid position 429 (e.g., E429A)⁶Amino acid position 446 (e.g., G446R)^(4,11) Amino acid position 453(e.g., S453Y)⁶ Amino acid position 454 (e.g., D454G)⁶ Amino acidposition 455 (e.g., K455N)⁴³ Amino acid position 456 (e.g., T456M^(3,6),T456K³⁵) Amino acid position 457 (e.g., G457G⁶, G457fs*6³³) Amino acidposition 469 (e.g., C469G)⁴¹ Amino acid position 478 (e.g., H478H)⁴¹Amino acid position 500 (e.g., Y500H)⁶ Amino acid position 525 (e.g.,R525X)⁴ Δ Amino acid position 529⁶ Amino acid position 535 (e.g.,H535L⁶, H535N⁴¹) Amino acid position 553 (e.g., P553P)⁴³ Amino acidposition 554 (e.g., D554N^(1,6), D554A³⁵) Δ Amino acid positions556-628⁴⁴ Δ Amino acid positions 559-563³⁵ Amino acid position 570(e.g., L570L)⁴¹ Amino acid position 577 (e.g., I577V)¹⁹ Amino acidposition 581 (e.g., E581K)³⁵ Amino acid positions 554 and 581 (e.g.,D554A + E581K)³⁵ Amino acid position 585 (e.g., E585X)²¹ Amino acidposition 600 (e.g., R600W^(2,4), R600Q⁶) Amino acid position 602 (e.g.,R602X)^(3,6) Amino acid position 628 (e.g., R628W)⁶ Amino acid position631 (e.g., R631Q)²⁸ Δ Amino acid positions 645-699⁴ Amino acid position661 (e.g., I661T)^(1,4,6) Amino acid position 665 (e.g., E665X)^(4,6)Amino acid position 672 (e.g., K672fs⁶, K672Vfs*1³⁵) Amino acid position674 (e.g., M674T)¹⁹ Amino acid positions 78 and 674 (e.g., H78Q/M674T)¹⁹Amino acid position 684 (e.g., D684D)⁴¹ Amino acid position 688 (e.g.,D688G)⁶ Amino acid position 694 (e.g., I694T⁶, I694N¹⁷) Amino acidposition 695 (e.g., E695K)²⁷ Amino acid position 709 (e.g., K709fs⁶,K709Qfs*41¹³) Amino acid position 717 (e.g., T717N)⁴ Amino acid position733 (e.g., G733R)⁶ Amino acid position 757 (e.g., Y757X)⁴ Amino acidposition 749 (e.g., L749P)²¹ Amino acid position 792 (e.g., P792fs)⁶ ΔAmino acid position 795-797⁶ Amino acid position 809 (e.g., I809L)²⁷Amino acid position 814 (e.g., K814N)²⁸ Amino acid position 833 (e.g.,R833Q²⁷, R833W⁴¹) Amino acid position 835 (e.g., K835Rfs*36)³⁵ Aminoacid position 845 (e.g., K845fs)²⁵ Amino acid position 849 (e.g.,R849Q)²⁴ Amino acid position 853 (e.g., F853S, F853fs)⁶ Amino acidposition 867 (e.g., R867C¹, R867fs⁶, R867H²³) Amino acid position 885(e.g., K885T)⁴¹ Amino acid position 888 (e.g., T888T)⁴¹ Amino acidposition 892 (e.g., G892R)⁶ Amino acid position 912 (e.g., G912R)³⁵Amino acid position 921 (e.g., S921S)⁴¹ Amino acid position 924 (e.g.,Y924C)²⁸ Amino acid position 930 (e.g., R930X⁶, R930Q²⁸) Amino acidposition 941 (e.g., R941X)³⁵ Amino acid position 946 (e.g., R946T)⁴¹Amino acid position 952 (e.g., R952Q^(5,9,15), R952X⁶) Amino acidposition 958 (e.g., N958fs)⁶ Amino acid position 960 (e.g., A960A)⁴¹ ΔAmino acid position 971⁴³ Amino acid position 976 (e.g., A976E⁴¹,A976A⁴³) Amino acid position 981 (e.g., E981K)²⁰ Amino acid position 994(e.g., S994R)⁴ Amino acid position 1011 (e.g., L1011fs*18)³³ Amino acidposition 1012 (e.g., S1012I)¹⁰ Amino acid position 1014 (e.g.,R1014X)^(6,11) Amino acid position 1015 (e.g., F1015L)²⁷ Amino acidposition 1023 (e.g., Q1023fs)⁶ Amino acid position 1040 (e.g.,G1040R)^(1,6) Amino acid position 1044 (e.g., S0144L)³⁴ Amino acidposition 1047 (e.g., L1047fs)⁶ Amino acid position 1050 (e.g., I1050K)³¹Amino acid position 1052 (e.g., L1052R)²⁸ Amino acid position 1095(e.g., W1095X)¹¹ Amino acid position 1098 (e.g., V1098X)³⁵ Amino acidposition 1131 (e.g., Q1131X)⁴⁴ Amino acid position 1142 (e.g.,A1142Tfs*35)⁴³ Amino acid position 1144 (e.g., Y1144Y)⁴³ Amino acidposition 1150 (e.g., I1150T)⁴¹ Amino acid position 1152 (e.g., A1152T)³⁰Amino acid position 1159 (e.g., P1159P)^(25,43) Amino acid position 1164(e.g., R1164X)⁶ Amino acid position 1193 (e.g., R1193fs*39)³³ Amino acidposition 1197 (e.g., V1197L)⁴¹ Amino acid position 1208 (e.g., A1208fs)⁶Amino acid position 1209 (e.g., Y1209Lfs*28)⁴ Amino acid position 1211(e.g., F1211L)²⁷ Amino acid position 1219 (e.g., D1219H⁵, D1219G²⁷)Amino acid position 1223 (e.g., S1223S)⁴¹ Amino acid position 1233(e.g., P1233P)⁴¹ Amino acid position 1241 (e.g., G1241fs)⁶ Amino acidposition 1248 (e.g., T1248T)⁴³ Splice site mutation IVS3 + 1_+3delGTG⁶Splice site mutation IVS3 − 2A > G⁶ IVS6 + 5T > G^(17,25) Splice sitemutation IVS8 + 1G > T⁶ IVS9 − G > A²⁶ IVS12 + 1G > A²⁵ Splice sitemutation IVS17 − 1G > A⁶ Splice site mutation IVS18 + 2T > C⁶ Splicesite mutation IVS20 − 4CT > AA Splice site mutation IVS21 + 5G > A⁶Splice site mutation IVS23 − 3C > A⁶ Splice site mutation IVS26 + 2T >A⁶ g.24774-42062del⁴ c.-4C > G⁴¹ c.145C > T¹² c.181 − 72G > A⁹ c.182 −5T > A⁴¹ c.182 − 72G > A⁴¹ c.246A > G⁹ c.239G > A³⁹ c.279 + 1_279 +3delGTG⁴⁶ c.280 − 2A > G⁴⁶ c.625_62715delinsACAGTAAT⁴⁶ c.554 + 122C > T⁹c.555 − 3T > C²⁷ c.625 + 5 G > T⁴ Amino acid position 209 (e.g., P209T)and c.625 + 5 G > T⁴ c.628 − 30G > A⁴¹ c.628 − 31C > T⁴¹ c.698 + 1G >T⁴⁶ c.698 + 20C > T⁴¹ c.782 − 1G > A⁴⁶ c.782 − 34G > A⁴¹ Δ795-797¹⁴c.782 − 1G > A⁴ c.852A > C²⁷ c.941 − 1G > A⁴⁶ c.1014C > T⁹ c.1029 +35G > A⁹ c.1221 − 8C · G⁴¹ 1226delA¹⁶ c.1429 + 1G > A⁴⁶ c.1429 + 2T >G¹³ c.1429 + 49G > A⁴¹ c.1430 − 42A > G⁴¹ c.1493T > C¹²c.1587_1589delCTT⁴⁶ c.1630 + 2T > G²⁷ c.1631 − 10T > A⁴¹ c.1637 − 37T >C⁴¹ 1660 G > A¹⁴ 1798 C > T¹⁴ 1799 G > A¹⁴ c.1819 − 39_41delAA⁹ c.1819 +1G > A³¹ c.1820 − 27G > A⁴¹ c.1918 + 8C > T²⁷ c.1933 − 1G > AK46c.2097 + 2T > C³² c.2097 + 60T > G⁴¹ c.2097 + 89T > C⁴¹ c.2097 + 97T >G⁴¹ c.2210 − 114T > C⁹ 2210delA¹⁶ c.2210 − 45_50dupATAAAA⁹ c.2285 + 29C· T⁴¹ c.2285 + 32A > G⁴¹ c.2286 − 4_2286-3delinsAA⁴⁶ c.2418 + 5G > A⁴⁶c.2707 + 3G > C²⁷ c.2707 + 9T > G⁴¹ c.2707 + 43A > G⁴¹ c.2709 − 59T >C⁴¹ c.2931 + 9A > G⁴¹ c.2931 + 59T > A⁴¹ c.2932 − 3C > A⁴⁶ c.2932 +59T > A⁹ c.2937A > C²⁷ c.3016 − 9C > A³¹ c.3033-3034del¹⁹3122delTCCTA/insACATCGATGTTGATGTTAGG⁴⁵ 3318 G > A¹⁴ c.3400 + 2T > A⁴⁶c.3401 − 175C > T⁹ c.3401 − 167C > T⁹ c.3401 − 108C > T⁹ c.3531 + 8G >T^(9,15) c.3532 − 15C > T⁹ Δ Phe ex 15⁴ Ex1_Ex13del⁶ Ex2_Ex6del³³Ex12_Ex14del²⁷ Skipped Exon 24⁴⁵ del5′UTR-ex18¹¹ c.*11C > T⁴¹ c.*1101 +366G > A⁷ g.92918del565³¹ GC preceding exon 16 (e.g., resulting in a 4bp deletion)⁴² Frameshift from the 5′ end of exon 16⁴² 5′ 1.4 kbdeletion⁴⁶

TABLE 2 Selected ATP8B1 Mutations Associated with PFIC-1 Amino acidposition 23 (e.g., P23L)⁵ Amino acid position 78 (e.g., H78Q)¹⁹ Aminoacid position 93 (e.g., A93A)⁶ Amino acid position 96 (e.g., A96G)²⁷Amino acid position 127 (e.g., L127P)⁶ Amino acid position 197 (e.g.,G197Lfs*10)²² Amino acid position 205 (e.g., N205fs)⁶ Amino acidposition 209 (e.g., P209T)⁴ Amino acid position 233 (e.g., G233R)³⁸Amino acid position 243 (e.g., L243fs*28)³³ Amino acid position 288(e.g., L288S)⁶ Amino acid position 296 (e.g., R296C)¹¹ Amino acidposition 308 (e.g., G308V^(1,6)) Amino acid position 320 (e.g.,M320Vfs*13)¹¹ Amino acid position 403 (e.g., S403Y)⁶ Amino acid position407 (e.g., S407N)⁴⁰ Amino acid position 412 (e.g., R412P)⁶ Amino acidposition 415 (e.g., Q415R)²⁷ Amino acid position 429 (e.g., E429A)⁶Amino acid position 446 (e.g., G446R)⁴ Amino acid position 456 (e.g.,T456M)^(3,6) Amino acid position 457 (e.g., G457G⁶, G457fs*6³³) Aminoacid position 500 (e.g., Y500H)⁶ Amino acid position 525 (e.g., R525X)⁴Δ Amino acid position 529⁶ Amino acid position 535 (e.g., H535L)⁶ Aminoacid position 554 (e.g., D554N)^(1,6) Amino acid position 577 (e.g.,I577V)¹⁹ Amino acid position 585 (e.g., E585X)²¹ Amino acid position 600(e.g., R600W)⁴ Amino acid position 602 (e.g., R602X)^(3,6) Amino acidposition 661 (e.g., I661T)^(4,6) Amino acid position 665 (e.g.,E665X)^(4,6) Δ Amino acid positions 645-699⁴ Amino acid position 672(e.g., K672fs)⁶ Amino acid position 674 (e.g., M674T)¹⁹ Amino acidpositions 78 and 674 (e.g., H78Q/M674T)¹⁹ Amino acid position 688 (e.g.,D688G)⁶ Amino acid position 694 (e.g., I694N)¹⁷ Amino acid position 695(e.g., E695K)²⁷ Amino acid position 709 (e.g., K709fs)⁶ Amino acidposition 717 (e.g., T717N)⁴ Amino acid position 733 (e.g., G733R)⁶ Aminoacid position 749 (e.g., L749P)²¹ Amino acid position 757 (e.g., Y757X)⁴Amino acid position 792 (e.g., P792fs)⁶ Amino acid position 809 (e.g.,I809L)²⁷ Amino acid position 853 (e.g., F853S, F853fs)⁶ Amino acidposition 867 (e.g., R867fs)⁶ Amino acid position 892 (e.g., G892R)⁶Amino acid position 930 (e.g., R930X⁶, R952Q¹⁵) Amino acid position 952(e.g., R952X)⁶ Amino acid position 958 (e.g., N958fs)⁶ Amino acidposition 981 (e.g., E981K)²⁰ Amino acid position 994 (e.g., S994R)⁴Amino acid position 1014 (e.g., R1014X)^(6,11) Amino acid position 1015(e.g., F1015L)²⁷ Amino acid position 1023 (e.g., Q1023fs)⁶ Amino acidposition 1040 (e.g., G1040R)^(1,6) Amino acid position 1047 (e.g.,L1047fs)⁶ Amino acid position 1095 (e.g., W1095X)¹¹ Amino acid position1208 (e.g., A1208fs)⁶ Amino acid position 1209 (e.g., Y1209Lfs*28)⁴Amino acid position 1211 (e.g., F1211L)²⁷ Amino acid position 1219(e.g., D1219H⁵, D1219G²⁷) Splice site mutation IVS3 + 1_+3delGTG⁶ Splicesite mutation IVS3 − 2A > G⁶ IVS6 + 5T > G¹⁷ Splice site mutation IVS8 +1G > T⁶ IVS9 − G > A²⁶ Splice site mutation IVS17 − 1G > A⁶ Splice sitemutation IVS18 + 2T > C⁶ Splice site mutation IVS21 + 5G > A⁶g.24774-42062del⁴ c.145C > T¹² c.239G > A³⁹ c.625 + 5 G > T⁴ Amino acidposition 209 (e.g., P209T) and c.625 + 5 G > T⁴ c.782 − 1G > A⁴c.1493T > C¹² c.1630 + 2T > G²⁷ 1660 G > A¹⁴ c.2707 + 3G > C²⁷ c.2097 +2T > C³² c.3033-3034del¹⁹ 3318 G > A¹⁴ c.3158 + 8G > T¹⁵ Δ Phe ex 15⁴Ex1_Ex13del⁶ Ex2_Ex6del³³ Ex12_Ex14del²⁷ del5′UTR-ex18¹¹ c.*1101 +366G > A⁷ GC preceding exon 16 (e.g., resulting in a 4 bp deletion)⁴²Frameshift from the 5′ end of exon 16⁴² ^(A) A mutation to ‘X’ denotesan early stop codon

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45 Jirsa et al., Hepatol Res. 2004, vol. 30(1), p. 1-3.

-   ⁴⁶ van der Woerd et al., Hepatology 2015, vol. 61(4), p. 1382-1391.

In some embodiments, the mutation in ATP8B1 is selected from L127P,G308V, T456M, D554N, F529del, I661T, E665X, R930X, R952X, R1014X, andG1040R.

TABLE 3 Exemplary ABCB11 Mutations Amino acid position 1 (e.g., M1V)⁹Amino acid position 4 (e.g., S4X)^(A,64) Amino acid position 8 (e.g.,R8X)⁸⁸ Amino acid position 19 (e.g., G19R)⁵⁶ Amino acid position 24(e.g., K24X)³⁵ Amino acid position 25 (e.g., S25X)^(5,14) Amino acidposition 26 (e.g., Y26Ifs*7)³⁸ Amino acid position 36 (e.g., D36D)²⁷Amino acid position 38 (e.g., K38Rfs*24)⁷³ Amino acid position 43 (e.g.,V43I)⁵⁷ Amino acid position 49 (e.g., Q49X)⁷³ Amino acid position 50(e.g., L50S, L50W)⁵⁷ Amino acid position 52 (e.g., R52W²⁶, R52R²⁸) Aminoacid position 56 (e.g., S56L)⁵⁸ Amino acid position 58 (e.g., D58N)⁶²Amino acid position 62 (e.g., M62K)⁹ Amino acid position 66 (e.g.,S66N)¹⁷ Amino acid position 68 (e.g., C68Y)⁴¹ Amino acid position 50(e.g., L50S)^(5,7) Amino acid position 71 (e.g., L71H)⁷³ Amino acidposition 74 (e.g., I74R)⁷¹ Amino acid position 77 (e.g., P77A)⁷³ Aminoacid position 87 (e.g., T87R)⁶⁷ Amino acid position 90 (e.g.,F90F)^(7,27) Amino acid position 93 (e.g., Y93S¹³, Y93X⁸⁸) Amino acidposition 96 (e.g., E96X)⁸⁸ Amino acid position 97 (e.g., L97X)³⁹ Aminoacid position 101 (e.g., Q101Dfs*8)⁹ Amino acid position 107 (e.g.,C107R)³⁶ Amino acid position 112 (e.g., I112T)⁹ Amino acid position 114(e.g., W114R)^(2,9) Amino acid position 123 (e.g. M123T)⁶⁷ Amino acidposition 127 (e.g., T127Hfs*6)⁵ Amino acid position 129 (e.g., C129Y)²⁵Amino acid position 130 (e.g., G130G)⁷⁷ Amino acid position 134 (e.g.,I134I)²⁸ Amino acid position 135 (e.g., E135K^(7,13), E135L¹⁷) Aminoacid position 137 (e.g., E137K)⁷ Amino acid position 157 (e.g., Y157C)⁵Amino acid position 161 (e.g., C161X)³⁹ Amino acid position 164 (e.g.,V164Gfs*7³⁰, V164I⁸⁵) Amino acid position 167 (e.g., A167S⁴, A167V⁷,A167T^(9,17)) Amino acid position 181 (e.g., R181I)³⁵ Amino acidposition 182 (e.g., I182K)⁹ Amino acid position 183 (e.g., M183V⁸,M183T⁹) Amino acid position 185 (e.g., M185I)⁷³ Amino acid position 186(e.g., E186G)^(2,7,22) Amino acid position 188 (e.g., G188W)⁷³ Aminoacid position 194 (e.g., S194P)⁷ Amino acid position 198 (e.g., L198P)⁷Amino acid position 199 (e.g., N199Ifs*15X)⁸⁸ Amino acid position 206(e.g., I206V)²⁸ Amino acid position 212 (e.g., A212T)⁷³ Amino acidposition 217 (e.g., M217R)⁸⁸ Amino acid position 225 (e.g., T225P)⁵⁷Amino acid position 226 (e.g., S226L)⁹ Amino acid position 232 (e.g.,L232Cfs*9)⁹ Amino acid position 233 (e.g., L233S)⁸⁶ Amino acid position238 (e.g., G238V)^(2,7) Amino acid position 242 (e.g., T242I)^(5,7)Amino acid position 245 (e.g., I245Tfs*26)⁵⁷ Amino acid position 256(e.g., A256G)⁹ Amino acid position 260 (e.g., G260D)⁷ Amino acidposition 269 (e.g., Y269Y)²⁷ Amino acid position 277 (e.g., A277E)⁷⁷Amino acid position 283 (e.g., E283D)⁷³ Amino acid positions 212 and 283(e.g., A212T + E283D)⁷³ Amino acid position 284 (e.g., V284L^(7,39),V284A⁷, V284D²³) Amino acid position 297 (e.g., E297G^(1,2,5,7), E297K⁷)Amino acid position 299 (e.g., R299K)²⁸ Amino acid position 303 (e.g.,R303K⁸, R303M⁶³ R303fsX321⁸³) Amino acid position 304 (e.g., Y304X)²⁶Amino acid position 312 (e.g., Q312H)⁷ Amino acid position 313 (e.g.,R313S)^(5,7) Amino acid position 314 (e.g., W314X)⁵⁷ Amino acid position318 (e.g., K318Rfs*26)²⁹ Amino acid position 319 (e.g., G319G)⁷ Aminoacid position 327 (e.g., G327E)^(5,7) Amino acid position 330 (e.g.,W330X)²⁴ Amino acid position 336 (e.g., C336S)^(2,7) Amino acid position337 (e.g., Y337H)^(21,27) Amino acid position 342 (e.g., W342G)⁵⁰ Aminoacid position 354 (e.g., R354X)⁹ Amino acid position 361 (e.g., Q361X⁵⁷,Q361R⁷⁴) Amino acid position 366 (e.g., V366V²⁸, V366D⁵⁷) Amino acidposition 368 (e.g., V368Rfs*27)⁵ Amino acid position 374 (e.g., G374S)³Amino acid position 380 (e.g., L380Wfs*18)⁵ Amino acid position 382(e.g., A382G)⁸⁸ Δ Amino acid positions 382-388⁵ Δ Amino acid positions383-389⁵⁷ Amino acid position 387 (e.g., R387H)⁹ Amino acid position 390(e.g., A390P)^(5,7) Amino acid position 395 (e.g., E395E)²⁸ Amino acidposition 404 (e.g., D404G)⁹ Amino acid position 410 (e.g., G410D)^(5,7)Amino acid position 413 (e.g., L413W)^(5,7) Amino acid position 415(e.g., R415X)⁴² Amino acid position 416 (e.g., I416I)²⁷ Amino acidposition 420 (e.g., I420T)⁹ Amino acid position 423 (e.g., H423R)¹³Amino acid position 432 (e.g., R432T)^(1,2,7) Amino acid position 436(e.g., K436N)⁴⁰ Amino acid position 440 (e.g., D440E)⁸⁸ Amino acidposition 444 (e.g., V444A)² Amino acid position 454 (e.g., V454X)⁴⁹Amino acid position 455 (e.g., G455E)⁹ Amino acid position 457 (e.g.,S457Vfs*23)⁸⁸ Amino acid position 461 (e.g., K461E)^(2,7) Amino acidposition 462 (e.g., S462R)⁸⁸ Amino acid position 463 (e.g., T463I)^(5,7)Amino acid position 466 (e.g., Q466K)^(5,7) Amino acid position 470(e.g., R470Q^(5,7), R470X⁹) Amino acid position 471 (e.g., Y472X)⁵ Aminoacid position 472 (e.g., Y472C^(5,27), Y472X¹⁴) Amino acid position 473(e.g., D473Q³⁵, D473V⁸⁸) Amino acid position 475 (e.g., C475X)²⁹ Aminoacid position 481 (e.g., V481E)^(5,7) Amino acid position 482 (e.g.,D482G)^(2,5,7) Amino acid position 484 (e.g., H484Rfs*5)⁹ Amino acidposition 487 (e.g., R487H², R487P⁵) Amino acid position 490 (e.g.,N490D)^(5,7) Amino acid position 493 (e.g., W493X)⁸ Amino acid positon496 (e.g., D496V)⁸⁸ Amino acid position 498 (e.g., I498T)^(2,7) Aminoacid position 499 (e.g., G499E)⁷³ Amino acid position 501 (e.g.,V501G)⁶⁸ Amino acid position 504 (e.g., E504K)⁷⁹ Amino acid position 510(e.g., T510T)⁷ Amino acid position 512 (e.g., I512T)^(5,7) Amino acidposition 515 (e.g., N515T^(5,7), N515D⁶⁴) Amino acid position 516 (e.g.,I516M)¹⁷ Amino acid position 517 (e.g., R517H)^(5,7) Amino acid position520 (e.g., R520X)⁵ Amino acid position 523 (e.g., A523G)¹³ Amino acidposition 528 (e.g., I528Sfs*21⁵, I528X⁹, I528T⁷³) Amino acid position535 (e.g., A535A⁷, A535X⁸⁹) Amino acid position 540 (e.g., F540L)⁴⁶Amino acid position 541 (e.g., I541L^(5,7), I541T^(5,17)) Amino acidposition 546 (e.g., Q546K³⁹, Q546H⁷³) Amino acid position 548 (e.g.,F548Y)^(5,7) Amino acid position 549 (e.g., D549V)⁹ Amino acid position554 (e.g., E554K)²¹ Amino acid position 556 (e.g., G556R)⁶⁷ Amino acidposition 558 (e.g., Q558H)²³ Amino acid position 559 (e.g., M559T)⁵⁷Amino acid position 562 (e.g., G562D^(5,7), G562S⁷³) Amino acid position570 (e.g., A570T^(2,5,7), A570V²⁶) Amino acid position 575 (e.g.,R575X^(2,5), R575Q²¹) Amino acid position 580 (e.g., L580P)⁵⁷ Amino acidposition 586 (e.g., T586I)⁷ Amino acid position 587 (e.g., S587X)⁷³Amino acid position 588 (e.g., A588V^(5,7), A588P⁷³) Amino acid position591 (e.g., N591S)^(2,7) Amino acid position 593 (e.g., S593R)^(2,7)Amino acid position 597 (e.g., V597V⁹, V597L¹³) Amino acid position 603(e.g., K603K)⁵⁵ Amino acid position 609 (e.g., H609Hfs*46)²⁶ Amino acidposition 610 (e.g., I610Gfs*45⁹, I610T⁵⁷)⁹ Amino acid position 615(e.g., H615R)²⁶ Amino acid position 616 (e.g., R616G²⁸, R616H⁷³) Aminoacid position 619 (e.g., T619A)²⁸ Amino acid position 623 (e.g.,A623A)²⁸ Amino acid position 625 (e.g., T625Nfs*5)²⁶ Amino acid position627 (e.g., I627T)⁷ Amino acid position 628 (e.g., G628Wfs*3)⁷⁰ Aminoacid position 636 (e.g., E636G)² Amino acid position 648 (e.g.,G648Vfs*6⁵, G648V⁵⁰) Amino acid position 655 (e.g., T655I)⁷ Amino acidposition 669 (e.g., I669V)²⁶ Amino acid position 676 (e.g., D676Y)¹¹Amino acid position 677 (e.g., M677V)^(7,13) Amino acid position 679(e.g., A679V)⁵⁸ Amino acid position 685 (e.g., G685W)⁶⁰ Amino acidposition 696 (e.g., R696W²⁷, R696Q⁵⁸) Amino acid position 698 (e.g.,R698H^(7,9), R698K⁶¹, R698C⁸⁸) Amino acid position 699 (e.g., S699P)⁹Amino acid position 701 (e.g., S701P)⁵⁸ Amino acid position 702 (e.g.,Q702X)⁸⁹ Amino acid position 709 (e.g., E709K)⁷ Amino acid position 710(e.g., P710P)⁷ Amino acid position 712 (e.g., L712L)²⁸ Amino acidposition 721 (e.g., Y721C)⁸⁸ Amino acid position 729 (e.g., D724N)³⁹Amino acid position 731 (e.g., P731S)²³ Amino acid position 740 (e.g.,P740Qfs*6)⁷³ Amino acid position 758 (e.g., G758R)⁵ Amino acid position766 (e.g., G766R)^(5,24) Amino acid position 772 (e.g., Y772X)⁵ Aminoacid position 804 (e.g., A804A)⁷ Amino acid position 806 (e.g., G806D⁴⁴,G806G⁵⁵) Amino acid position 809 (e.g., S809F)⁸¹ Amino acid position 817(e.g., G817G)⁸⁸ Amino acid position 818 (e.g., Y818F)⁷ Amino acidposition 824 (e.g., G824E)⁴² Amino acid position 825 (e.g., G825G)⁷³Amino acid position 830 (e.g., R830Gfs*28)⁷³ Amino acid position 832(e.g., R832C^(7,26), R832H⁴¹) Amino acid position 842 (e.g., D842G)²Amino acid position 848 (e.g., D848N)⁷³ Amino acid position 855 (e.g.,G855R)¹¹ Amino acid position 859 (e.g., T859R)^(5,7) Amino acid position865 (e.g., A865V)²⁷ Amino acid position 866 (e.g., S866A)⁵⁷ Amino acidposition 868 (e.g., V868D)⁷³ Amino acid position 869 (e.g., Q869P)⁷³Amino acid position 875 (e.g., Q875X)⁷³ Amino acid position 877 (e.g.,G877R)⁵⁶ Amino acid position 879 (e.g., I879R)⁸⁸ Amino acid position 893(e.g., A893V)⁵⁷ Amino acid position 901 (e.g., S901R¹⁷, S901I⁷³) Aminoacid position 903 (e.g., V903G)⁵⁷ Δ Amino acid position 919¹² Amino acidposition 923 (e.g., T923P)^(2,7) Amino acid position 926 (e.g.,A926P)^(2,7) Amino acid position 928 (e.g., R928X¹⁵, R928Q⁴⁰) Amino acidposition 930 (e.g., K930X⁵, K930Efs*79^(5,10), K930Efs*49²⁶) Amino acidposition 931 (e.g., Q931P)²⁷ Amino acid position 945 (e.g., S945N)⁵⁷Amino acid position 948 (e.g., R948C)^(5,7,26) Amino acid position 958(e.g., R958Q)²⁸ Amino acid position 969 (e.g., K969K)⁸⁸ Δ Amino acidpositions 969-972⁵ Amino acid position 973 (e.g., T973I)⁵⁷ Amino acidposition 976 (e.g., Q976R⁵⁸, Q976X⁸⁸) Amino acid position 979 (e.g.,N979D)^(5,7) Amino acid position 981 (e.g., Y981Y)²⁸ Amino acid position982 (e.g., G982R)^(2,5,7) Amino acid positions 444 and 982 (e.g.,V444A + G982R)³⁸ Amino acid position 995 (e.g., A995A)²⁸ Amino acidposition 1001 (e.g., R1001R)⁹ Amino acid position 1003 (e.g., G1003R)²⁴Amino acid position 1004 (e.g., G1004D)^(2,7) Amino acid position 1027(e.g., S1027R)²⁶ Amino acid position 1028 (e.g., A1028A^(7,10,88),A1028E⁸⁸) Amino acid position 1029 (e.g., T1029K)⁵ Amino acid position1032 (e.g., G1032R)¹² Amino acid position 1041 (e.g., Y1041X)⁹ Aminoacid position 1044 (e.g., A1044P)⁸⁸ Amino acid position 1050 (e.g.,R1050C)^(2,7,57) Amino acid position 1053 (e.g., Q1053X)⁵⁷ Amino acidposition 1055 (e.g., L1055P)³⁶ Amino acid position 1057 (e.g., R1057X²,R1057Q⁵⁸) Amino acid position 1058 (e.g., Q1058Hfs*38⁹, Q1058fs*38¹⁷,Q1058X⁷³) Amino acid position 1061 (e.g., I1061Vfs*34)⁹ Amino acidposition 1083 (e.g., C1083Y)⁴⁷ Amino acid position 1086 (e.g., T1086T)²⁸Amino acid position 1090 (e.g., R1090X)^(2,5) Amino acid position 1099(e.g., L1099Lfs*38)²⁶ Amino acid position 1100 (e.g., S1100Qfs*38)¹³Amino acid position 1110 (e.g., A1110E)^(5,7) Amino acid position 1112(e.g., V1112F)⁷⁰ Amino acid position 1116 (e.g., G1116R⁷, G1116F^(9,17),G1116E³⁶) Amino acid position 1120 (e.g., S1120N)⁸⁸ Amino acid position1128 (e.g., R1128H^(2,7), R1128C^(5,7,13)) Amino acid position 1131(e.g., D1131V)²⁷ Amino acid position 1144 (e.g., S1144R)⁷ Amino acidposition 1147 (e.g., V1147X)⁵ Amino acid position 1153 (e.g.,R1153C^(2,5,7), R1153H⁵) Amino acid position 1154 (e.g., S1154P)^(5,7)Amino acid position 1162 (e.g., E1162X)³⁹ Δ Amino acid position 1165⁸⁸Amino acid position 1164 (e.g., V1164Gfs*7) Amino acid position 1173(e.g., N1173D)⁵⁷ Amino acid position 1175 (e.g., K1175T)⁵⁸ Amino acidposition 1186 (e.g., E1186K)⁷ Amino acid position 1192 (e.g.,A1192Efs*50)⁹ Amino acid position 1196 (e.g., Q1196X)⁸⁸ Amino acidposition 1197 (e.g., L1197G)⁷ Amino acid position 1198 (e.g., H1198R)²⁷Amino acid position 1204 (e.g., L1204P)⁸⁸ Amino acid position 1208 (e.g.Y1208C)⁷³ Amino acid position 1210 (e.g., T1210P^(5,7), T1210F⁵⁷) Aminoacid position 1211 (e.g., N1211D)⁷ Amino acid position 1212 (e.g.,V1212F)³⁶ Amino acid position 1215 (e.g., Q1215X)⁵ Amino acid position1221 (e.g., R1221K)⁵³ Amino acid position 1223 (e.g., E1223D)⁷ Aminoacid position 1226 (e.g., R1226P)⁷³ Amino acid position 1228 (e.g.,A1228V)⁷ Amino acid position 1231 (e.g., R1231W^(5,7), R1231Q^(5,7))Amino acid position 1232 (e.g., A1232D)¹⁷ Amino acid position 1235(e.g., R1235X)^(5,12) Amino acid position 1242 (e.g., L1242I)^(5,7)Amino acid position 1243 (e.g., D1243G)⁶⁷ Amino acid position 1249(e.g., L1249X)⁷³ Amino acid position 1256 (e.g., T1256fs*1296)⁸³ Aminoacid position 1268 (e.g., R1268Q)^(2,7) Amino acid position 1276 (e.g.,R1276H)³⁰ Amino acid position 1283 (e.g., A1283A²⁸, A1283V⁸⁸) Amino acidposition 1292 (e.g., G1292V)⁷³ Amino acid position 1298 (e.g., G1298R)⁵Amino acid position 1302 (e.g., E1302X)⁵ Amino acid position 1311 (e.g.,Y1311X)⁵⁷ Amino acid position 1316 (e.g., T1316Lfs*64)¹⁵ Amino acidposition 1321 (e.g., S1321N)⁵⁷ Intron 4 ((+3)A > C)¹ IVS4 − 74A > T⁸⁹Splice site mutation 3′ Intron 5 c.3901G > A⁵ Splice site mutation 5;Intron 7 c.6111G > A⁵ Splice site mutation IVS7 + 1G > A¹⁴ IVS7 + 5G >A⁴⁰ IVS8 + 1G > C⁷⁶ Splice site mutation 5′ Intron 9 c.9081delG⁵ Splicesite mutation 5′ Intron 9 c.9081G > T⁵ Splice site mutation 5′ Intron 9c.9081G > A⁵ Splice site mutation IVS9 + 1G > T¹⁴ Splice site mutation3′ Intron 13 c.143513_1435 − 8del⁵ Splice site mutation IVS13del −13{circumflex over ( )} − 8¹⁴ Splice site mutation 3′ Intron 16c.20128T > G⁵ Splice site mutation IVS16 − 8T > G¹⁴ Splice site mutation5′ Intron 18 c.21781G > T⁵ Splice site mutation 5′ Intron 18 c.21781G >A⁵ Splice site mutation 5′ Intron 18 c.21781G > C⁵ Splice site mutation3′ Intron 18 c.21792A > G⁵ Splice site mutation IVS18 + 1G > A¹⁴ Splicesite mutation 5′ Intron 19 c.2343 + 1G > T⁵ Splice site mutation 5′Intron 19 c.2343 + 2T > C⁵ Splice site mutation IVS19 + 2T > C¹⁴ Splicesite mutation IVS19 + 1G > A²² Splice site mutation 3′ Intron 21c.26112A > T⁵ IVS22 + 3A > G⁸⁹ IVS 23 − 8 G − A³⁶ IVS24 + 5G > A⁵¹Splice site mutation 5′ Intron 24 c.32131delG⁵ IVS35 − 6C > G⁸⁹ Putativesplice mutation 1198 − 1G > C¹⁷ Putative splice mutation 1810 − 3C > G¹⁷Putative splice mutation 2178 + 1G > A¹⁷ Putative splice mutation 2344 −1G > T¹⁷ Putative splice mutation c.2611 − 2A > T³⁹ Putative splicemutation 3213 + 1_3213 + 2delinsA¹⁷ c.−24C > A^(44,78) c.76 13 G > T⁹c.77 − 19T > A⁵² c.90_93delGAAA¹⁸ c.124G > A⁶⁹ c.150 + 3 A > C¹⁰ 174C >T⁵⁴ c.245T > C⁸⁷ c.249_250insT¹⁸ 270T > C⁵⁴ 402C > T⁵⁴ 585G > C⁵⁴c.611 + 1G > A⁷⁰ c.611 + 4A > G³⁶ c.612 − 15_−6del10bp⁵⁵ c.625A > C³¹c.627 + 5G > T³¹ c.625A > C/c.627 + 5G > T³¹ 696G > T⁵⁴ c. 784 + 1G >C⁴⁹ 807T > C⁵⁴ c.886C > T³¹ c.890A > G⁵⁹ c.908 + 1G > A⁵⁷ c.908 + 5G >A⁵⁵ c.908delG⁵⁹ c.909 − 15A > G⁶⁶ 957A > G⁵⁴ c.1084 − 2A > G⁵⁷ 1145 1 bpdeletion⁹⁰ 1281C > T^(54,57) c.1309 − 165C > T¹⁹ c.1434 + 174G > A¹⁹c.1434 + 70C > T¹⁹ c.1530C > A⁵⁷ c.1587-1589delCTT³¹ c.1621A > C^(33,59)c.1638 + 32T > C⁶⁶ c.1638 + 80C > T⁶⁶ 1671C > T⁵⁴ 1791G > T⁵⁴ 1939delA¹⁴c.2075 + 3A > G⁵³ c.2081T > A³¹ c.2093G > A⁶⁵ 2098delA¹⁶ c.2138 − 8T >G⁶⁷ 2142A > G⁵⁴ c.2178 + 1G > T^(36,39) c.2179 − 17C > A⁶⁶ c.2344 −157T > G⁶⁶ c.2344 − 17T > C⁶⁶ c.2417G > A⁷⁸ c.2541delG⁸⁷ c.2620C >T^(32,33) c.2815 − 8A > G⁵⁵ c.3003A > G³⁷ c.3084A > G^(48,54) c.3213 + 4A > G^(9,37) c.3213 + 5 G > A⁹ c.3268C > T⁷⁵ 3285A > G⁵⁴ c.3382C > T⁷⁵3435A > G⁵⁴ c.3491delT⁷² c.3589C > T⁵⁷ c.3765(+1 + 5)del5⁴² c.3766 −34A > G⁶⁶ c.3767 − 3768insC⁶ c.3770delA⁶⁷ c.3826C > T⁷² c.3846C > T⁵⁷c.3929delG⁶⁷ c.*236A > G⁶⁶ 1145delC⁸ Ex13_Ex17del⁸²

TABLE 4 Selected ABCB11 Mutations Associated with PFIC-2 Amino acidposition 1 (e.g., M1V)⁹ Amino acid position 4 (e.g., S4X)⁶⁴ Amino acidposition 19 (e.g., G19R)⁵⁶ Amino acid position 25 (e.g., S25X)¹⁴ Aminoacid position 26 (e.g., Y26Ifs*7)³⁸ Amino acid position 50 (e.g.,L50S)^(7,57) Amino acid position 52 (e.g., R52W)²⁶ Amino acid position58 (e.g., D58N)⁶² Amino acid position 62 (e.g., M62K)⁹ Amino acidposition 66 (e.g., S66N)¹⁷ Amino acid position 68 (e.g., C68Y)⁴¹ Aminoacid position 93 (e.g., Y93S)¹³ Amino acid position 101 (e.g.,Q101Dfs*8)⁹ Amino acid position 107 (e.g., C107R)³⁶ Amino acid position112 (e.g., I112T)⁹ Amino acid position 114 (e.g., W114R)^(2,9) Aminoacid position 129 (e.g., C129Y)²⁵ Amino acid position 135 (e.g.,E135K¹³, E135L¹⁷) Amino acid position 167 (e.g., A167V⁷, A167T^(9,17))Amino acid position 182 (e.g., I182K)⁹ Amino acid position 183 (e.g.,M183V⁸, M183T⁹) Amino acid position 225 (e.g., T225P)⁵⁷ Amino acidposition 226 (e.g., S226L)⁹ Amino acid position 232 (e.g., L232Cfs*9)⁹Amino acid position 233 (e.g., L233S)⁸⁶ Amino acid position 238 (e.g.,G238V)^(2,7) Amino acid position 242 (e.g., T242I)⁷ Amino acid position245 (e.g., I245Tfs*26)⁵⁷ Amino acid position 256 (e.g., A256G)⁹ Aminoacid position 260 (e.g., G260D)⁵⁷ Amino acid position 284 (e.g., V284L)⁷Amino acid position 297 (e.g., E297G)^(2,7) Amino acid position 303(e.g., R303K⁸, R303M⁶³, R303fsX321⁸³) Amino acid position 304 (e.g.,Y304X)²⁶ Amino acid position 312 (e.g., Q312H)⁷ Amino acid position 313(e.g., R313S)⁷ Amino acid position 314 (e.g., W314X)⁵⁷ Amino acidposition 318 (e.g., K318Rfs*26)²⁹ Amino acid position 327 (e.g., G327E)⁷Amino acid position 330 (e.g., V330X)²⁴ Amino acid position 336 (e.g.,C336S)^(2,7) Amino acid position 337 (e.g., Y337H)²¹ Amino acid position342 (e.g., W342G)⁵⁰ Amino acid position 354 (e.g., R354X)⁹ Amino acidposition 361 (e.g., Q361X)⁵⁷ Amino acid position 366 (e.g., V366D)⁵⁷Amino acid position 386 (e.g., G386X)³⁴ Δ Amino acid positions 383-389⁵⁷Amino acid position 387 (e.g., R387H)⁹ Amino acid position 390 (e.g.,A390P)⁷ Amino acid position 410 (e.g., G410D)⁷ Amino acid position 413(e.g., L413W)⁷ Amino acid position 415 (e.g., R415X)⁴² Amino acidposition 420 (e.g., I420T)⁹ Amino acid position 454 (e.g., V454X)⁴⁹Amino acid position 455 (e.g., G455E)⁹ Amino acid position 461 (e.g.,K461E)^(2,7) Amino acid position 463 (e.g., T463I)⁷ Amino acid position466 (e.g., Q466K)⁷ Amino acid position 470 (e.g., R470Q⁷, R470X⁹) Aminoacid position 472 (e.g., Y472X¹⁴, Y472C²⁷) Amino acid position 475(e.g., C475X)²⁹ Amino acid position 481 (e.g., V481E)⁷ Amino acidposition 482 (e.g., D482G)^(2,7) Amino acid position 484 (e.g.,H484Rfs*5)⁹ Amino acid position 487 (e.g., R487H², R487P⁸⁴) Amino acidposition 490 (e.g., N490D)⁷ Amino acid position 493 (e.g., W493X)⁸ Aminoacid position 498 (e.g., I498T)⁷ Amino acid position 501 (e.g., V501G)⁶⁸Amino acid position 512 (e.g., I512T)⁷ Amino acid position 515 (e.g.,N515T⁷, N515D⁶⁴) Amino acid position 516 (e.g., I516M)¹⁷ Amino acidposition 517 (e.g., R517H)⁷ Amino acid position 520 (e.g., R520X)⁵⁷Amino acid position 523 (e.g., A523G)¹³ Amino acid position 528 (e.g.,I528X)⁹ Amino acid position 540 (e.g., F540L)⁴⁶ Amino acid position 541(e.g., I541L⁷, I541T¹⁷) Amino acid position 548 (e.g., F548Y)⁷ Aminoacid position 549 (e.g., D549V)⁹ Amino acid position 554 (e.g., E554K)²¹Amino acid position 559 (e.g., M559T)⁵⁷ Amino acid position 562 (e.g.,G562D)⁷ Amino acid position 570 (e.g., A570T⁷, A570V²⁶) Amino acidposition 575 (e.g., R575X², R575Q²¹) Amino acid position 588 (e.g.,A588V)⁷ Amino acid position 591 (e.g., N591S)^(9,17) Amino acid position593 (e.g., S593R)^(2,7) Amino acid position 597 (e.g., V597V⁹, V597L¹³)Amino acid positions 591 and 597 (e.g., N591S + V597V)⁹ Amino acidposition 603 (e.g., K603K)⁵⁵ Amino acid position 609 (e.g.,H609Hfs*46)²⁶ Amino acid position 610 (e.g., I610Gfs*45)⁹ Amino acidposition 615 (e.g., H615R)²⁶ Amino acid position 625 (e.g., T625Nfs*5)²⁶Amino acid position 627 (e.g., I627T)⁷ Amino acid position 636 (e.g.,E636G)² Amino acid position 669 (e.g., I669V)²⁶ Amino acid position 698(e.g., R609H)⁹ Amino acid positions 112 and 698 (e.g., I112T + R698H)⁹Amino acid position 699 (e.g., S699P)⁹ Amino acid position 766 (e.g.,G766R)²⁴ Amino acid position 806 (e.g., G806G)⁵⁵ Amino acid position 824(e.g., G824E)⁴² Amino acid position 832 (e.g., R832C^(7,26), R832H⁴¹)Amino acid position 842 (e.g., D842G)² Amino acid position 859 (e.g.,T859R)⁷ Amino acid position 865 (e.g., A865V)⁴⁵ Amino acid position 877(e.g., G877R)⁵⁶ Amino acid position 893 (e.g., A893V)⁵⁷ Amino acidposition 901 (e.g., S901R)¹⁷ Amino acid position 903 (e.g., V903G)⁵⁷ ΔAmino acid position 919¹² Amino acid position 928 (e.g., R928X)^(15,21)Amino acid position 930 (e.g., K930Efs*79¹⁰, K930Efs*49²⁶) Amino acidposition 948 (e.g., R948C)^(7,26) Amino acid position 979 (e.g., N979D)⁷Amino acid position 982 (e.g., G982R)^(2,7) Amino acid positions 444 and982 (e.g., V444A + G982R)³⁸ Amino acid position 1001 (e.g., R1001R)⁹Amino acid position 1003 (e.g., G1003R)²⁴ Amino acid position 1004(e.g., G1004D)^(2,7) Amino acid position 1027 (e.g., S1027R)²⁶ Aminoacid position 1028 (e.g., A1028A)¹⁰ Amino acid position 1032 (e.g.,G1032R)¹² Amino acid position 1041 (e.g., Y1041X)⁹ Amino acid position1050 (e.g., R1050C)⁵⁷ Amino acid position 1053 (e.g., Q1053X)⁵⁷ Aminoacid position 1055 (e.g., L1055P)³⁶ Amino acid position 1057 (e.g.,R1057X)² Amino acid position 1058 (e.g., Q1058Hfs*38⁹, Q1058fs*38¹⁷)Amino acid position 1061 (e.g., I1061Vfs*34)⁹ Amino acid position 1083(e.g., C1083Y)⁴⁷ Amino acid position 1090 (e.g., R1090X)² Amino acidposition 1099 (e.g., L1099Lfs*38)²⁶ Amino acid position 1100 (e.g.,S1100Qfs*38)¹³ Amino acid position 1110 (e.g., A1110E)⁷ Amino acidposition 1116 (e.g., G1116R⁷, G1116F^(9,17), G1116E³⁶) Amino acidposition 1128 (e.g., R1128C)^(7,13) Amino acid position 1131 (e.g.,D1131V)²⁷ Amino acid position 1144 (e.g., S1144R)⁷ Amino acid position1153 (e.g., R1153C^(2,7), R1153H^(7,26)) Amino acid position 1154 (e.g.,S1154P)⁷ Amino acid position 1173 (e.g., N1173D)⁵⁷ Amino acid position1192 (e.g., A1192Efs*50)⁹ Amino acid position 1198 (e.g., H1198R)²⁷Amino acid position 1210 (e.g., T1210P⁷, T1210F⁵⁷) Amino acid position1211 (e.g., N1211D)⁷ Amino acid position 1212 (e.g., V1212F)³⁶ Aminoacid position 1231 (e.g., R1231W⁷, R1223Q⁷) Amino acid position 1232(e.g., A1232D)¹⁷ Amino acid position 1235 (e.g., R1235X)¹² Amino acidposition 1242 (e.g., L1242I)⁷ Amino acid position 1256 (e.g.,T1256fs*1296)⁸³ Amino acid position 1268 (e.g., R1268Q)^(2,7) Amino acidposition 1302 (e.g. E1302X)⁵⁷ Amino acid position 1311 (e.g., Y1311X)⁵⁷Amino acid position 1316 (e.g., T1316Lfs*64)¹⁵ Intron 4 ((+3)A > C)¹Splice site mutation IVS7 + 1G > A¹⁴ IVS8 + 1G > C⁷⁶ Splice sitemutation IVS9 + 1G > T¹⁴ Splice site mutation IVS13del-13{circumflexover ( )}-8¹⁴ Splice site mutation IVS16 − 8T > G¹⁴ Splice site mutationIVS18 + 1G > A¹⁴ Splice site mutation IVS19 + 2T > C¹⁴ IVS 23 − 8 G −A³⁶ IVS24 + 5G > A⁵¹ Putative splice mutation 1198 − 1G > C¹⁷ Putativesplice mutation 1810 − 3C > G¹⁷ Putative splice mutation 2178 + 1G > A¹⁷Putative splice mutation 2344 − 1G > T¹⁷ Putative splice mutation 3213 +1_3213 + 2delinsA¹⁷ c.-24C > A⁷⁸ c.76 13 G > T⁹ c.77 − 19T > A⁵²c.90_93delGAAA¹⁸ c.124G > A⁶⁹ c.150 + 3 A > C¹⁰ c.249_250insT¹⁸ c.611 +1G > A⁸⁴ c.611 + 4A > G³⁶ c.612 − 15_-6del10bp⁵⁵ c.625A > C³¹ c.627 +5G > T³¹ c.625A > C/c.627 + 5G > T³¹ c.886C > T³¹ c.890A > G⁵⁹ c.908 +1G > A⁵⁷ c.908 + 5G > A⁵⁵ c.908delG⁵⁹ 1273 1 bp deletion⁹¹ c.1084 − 2A >G⁵⁷ c.1445A > G⁵⁹ c.1587-1589delCTT³¹ c.1621A > C⁵⁹ 1939delA¹⁴ c.2081T >A³¹ 2098delA¹⁶ c.2343 + 1 G > T⁸⁰ c.2178 + 1G > T³⁶ c.2417G > A⁷⁸c.2620C > T³² c.2815 − 8A > G⁵⁵ c.3003A > G³⁷ c.3213 + 4 A > G^(9,37)c.3213 + 5 G > A⁹ c.3268C > T⁷⁵ c.3382C > T⁷⁵ c.3765(+1 + 5)del5⁴²c.3767-3768insC⁶ 1145delC⁸ Ex13_Ex17del⁸² ^(A) A mutation to ‘X’ denotesan early stop codon

REFERENCES FOR TABLES 3 AND 4

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In some embodiments, the mutation in ABCB11 is selected from A167T,G238V, V284L, E297G, R470Q, R470X, D482G, R487H, A570T, N5915, A865V,G982R, R1153C, and R1268Q.

Provided are methods of treating PFIC (e.g., PFIC-1 and PFIC-2) in asubject that includes performing an assay on a sample obtained from thesubject to determine whether the subject has a mutation associated withPFIC (e.g., a ATP8B1, ABCB11, ABCB4, TJP2, NR1H4 or Myo5b mutation), andadministering (e.g., specifically or selectively administering) atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof, to the subject determined tohave a mutation associated with PFIC. In some embodiments, the mutationis an ATP8B1 or ABCB11 mutation. For example, a mutation as provided inany one of Tables 1-4. In some embodiments, the mutation in ATP8B1 isselected from L127P, G308V, T456M, D554N, F529del, I661T, E665X, R930X,R952X, R1014X, and G1040R. In some embodiments, the mutation in ABCB11is selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H,A570T, N5915, A865V, G982R, R1153C, and R1268Q.

Also provided are methods for treating PFIC (e.g., PFIC-1 and PFIC-2) ina subject in need thereof, the method comprising: (a) detecting amutation associated with PFIC (e.g., a ATP8B1, ABCB11, ABCB4, TJP2,NR1H4 or Myo5b mutation) in the subject; and (b) administering to thesubject a therapeutically effective amount of the formulation disclosedherein. In some embodiments, methods for treating PFIC can includeadministering a therapeutically effective amount of a compound offormula (I), or a pharmaceutically acceptable salt thereof, to a subjecthaving a mutation associated with PFIC (e.g., an ATP8B1, ABCB11, ABCB4,TJP2, NR1H4 or Myo5b mutation). In some embodiments, the mutation is anATP8B1 or ABCB11 mutation. For example, a mutation as provided in anyone of Tables 1-4. In some embodiments, the mutation in ATP8B1 isselected from L127P, G308V, T456M, D554N, F529del, I661T, E665X, R930X,R952X, R1014X, and G1040R. In some embodiments, the mutation in ABCB11is selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H,A570T, N5915, A865V, G982R, R1153C, and R1268Q.

In some embodiments, the subject is determined to have a mutationassociated with PFIC in a subject or a biopsy sample from the subjectthrough the use of any art recognized tests, including next generationsequencing (NGS). In some embodiments, the subject is determined to havea mutation associated with PFIC using a regulatory agency-approved,e.g., FDA-approved test or assay for identifying a mutation associatedwith PFIC in a subject or a biopsy sample from the subject or byperforming any of the non-limiting examples of assays described herein.Additional methods of diagnosing PFIC are described in Gunaydin, M. etal., Hepat Med. 2018, vol. 10, p. 95-104, incorporated by reference inits entirety herein.

In some embodiments, the treatment of PFIC (e.g., PFIC-1 or PFIC-2)decreases the level of serum bile acids in the subject. In someembodiments, the level of serum bile acids is determined by, forexample, an ELISA enzymatic assay or the assays for the measurement oftotal bile acids as described in Danese et al., PLoS One. 2017, vol.12(6): e0179200, which is incorporated by reference herein in itsentirety. In some embodiments, the level of serum bile acids candecrease by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to70%, 50% to 80%, or by more than 90% of the level of serum bile acidsprior to administration of the formulation disclosed herein. In someembodiments, the treatment of PFIC includes treatment of pruritus.

In another aspect, the invention relates to a process for thepreparation of the pharmaceutical formulation as disclosed herein,comprising the step of preparing a homogeneous aqueous suspension ofodevixibat. In a preferred embodiment, the coating suspension isprepared by dispersing odevixibat in water by wet milling.

In a more specific embodiment, the process comprises the steps of:

-   -   a) wetting odevixibat in water using a homogenizer; and    -   b) dispersing the wetted odevixibat in water by wet milling,        thereby obtaining a homogeneous aqueous suspension of        odevixibat.

In some embodiments, odevixibat is sieved prior to the wetting of stepa).

In some embodiments, the process further comprises the step of adding afilm-forming polymer to the suspension. A film-forming polymer canfacilitate a uniform dispersion of odevixibat in the suspension. Thefilm-forming polymer may be added either before or after the wet millingstep. In some embodiments, the wet milling is more effective in theabsence of the film-forming polymer. In a preferred embodiment,therefore, the film-forming polymer is added to the homogeneous aqueoussuspension of odevixibat obtained in step (b).

The homogeneity of the coating suspension may be checked either beforeor after addition of the film-forming polymer. When the size of theodevixibat agglomerates can be determined by optical microscopy, such asdescribed in the experimental section, the coating suspension should notcontain agglomerates larger than 200 μm. The suspension preferably doesnot contain agglomerates larger than 100 μm, and more preferably doesnot contain agglomerates larger than 50 μm. When the size of theagglomerates is determined by light scattering techniques, such asLALLS, the d₉₀ value for the particle size distribution of the coatingsuspension is preferably smaller than 15 μm, such as smaller than 14 μm,such as smaller than 13 μm, such as smaller than 12 μm, such as smallerthan 11 μm, or such as smaller than 10 μm.

In another preferred embodiment, the coating suspension does not containsurfactants.

In another aspect, the invention relates to the formulation obtained byany of the processes disclosed herein.

Definitions

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions and/or dosage forms that are suitablefor human pharmaceutical use and that are generally safe, non-toxic andneither biologically nor otherwise undesirable.

As used herein, the term “about” refers to a value or parameter hereinthat includes (and describes) embodiments that are directed to thatvalue or parameter per se. For example, description referring to “about20” includes description of “20.” Numeric ranges are inclusive of thenumbers defining the range. Generally speaking, the term “about” refersto the indicated value of the variable and to all values of the variablethat are within the experimental error of the indicated value (e.g.,within the 95% confidence interval for the mean) or within 10 percent ofthe indicated value, whichever is greater.

The term “crystal modification” refers to a crystalline solid phase ofan organic compound. A crystal modification can be either a solvate oran an solvate.

The term “solvate” refers to a crystalline solid phase of an organiccompound, which has solvent (i.e., solvent molecules) incorporated intoits crystal structure. A “hydrate” is a solvate wherein the solvent iswater.

The term “sesquihydrate” refers to a hydrate containing about 1.5 molesof water associated with the crystal per mole of organic compound (i.e.,a 1.5 hydrate). As used herein, a sesquihydrate includes from about 1.2to about 1.8, more preferably from about 1.3 to about 1.7, morepreferably from about 1.4 to about 1.6 and even more preferably fromabout 1.45 to about 1.55 moles of water associated with each mole ofodevixibat in a crystal. The amount of water calculated herein excludeswater adsorbed to the surface of the crystal.

The term “mixed solvate” refers to a crystalline solid phase of anorganic compound, which has two or more different solvent moleculesincorporated into its crystal structure. One of the at least two solventmolecules may be water.

The term “slurry” refers to a saturated solution to which an excess ofsolid is added, thereby forming a mixture of solid and saturatedsolution.

As used herein, the term “void volumes” refers to channels, layers orother more or less isolated voids in the crystal structure.

The crystallinity of a crystalline sample of odevixibat may be measurede.g. by X-Ray Powder Diffraction (XRPD) methods or by DifferentialScanning calorimetry (DSC) methods, such as the method disclosed in theexperimental section. When reference is made herein to a crystallinecompound, preferably the crystallinity as measured by DSC methods isgreater than about 70%, such as greater than about 80%, particularlygreater than about 90%, more particularly greater than about 95%. Insome embodiments, the degree of crystallinity as measured by DSC methodsis greater than about 98%. In some embodiments, the degree ofcrystallinity as measured by DSC methods is greater than about 99%. The% crystallinity refers to the percentage by weight of the total samplemass which is crystalline.

The invention will now be described by the following examples which donot limit the invention in any respect. All cited documents andreferences mentioned herein are incorporated by reference in theirentireties.

Abbreviations

DMF dimethylformamide

DMSO dimethyl sulfoxide

EtOH ethanol

MeOH methanol

RH relative humidity

2-PrOH 2-propanol

Experimental Methods

X-Ray Powder Diffraction (XRPD) Analysis

Analyses were performed at 22° C. on a PANalytical X'Pert Prodiffractometer equipped with a Cu long fine focus X-ray tube and aPIXcel detector. Automatic divergence and anti-scatter slits were usedtogether with 0.02 rad Soller slits and a Ni-filter. Dry samples weresmeared onto cut Silicon Zero Background Holders (ZBH) and analysedbetween 2-40° in 2-theta with an analysis time of 17 minutes. All slurrysamples were dripped on tempered porous Alumina filter substrates andanalysed twice as they dried, first with a one minute 16-second scan(2-30° in 2-theta) and then a 7-minute scan (2-30° in 2-theta). A final17-minute scan was performed when the sample had dried for severalhours.

The samples were spun during analysis in order to increase therandomness of the samples. The following experimental settings wereused:

Tube tension and current: 40 kV, 50 mA

Wavelength alpha1 (CuKα1): 1.5406 Å

Wavelength alpha1 (CuKα2): 1.5444 Å

Wavelength alpha1 and alpha1 mean (CuKα): 1.5418 Å

It is known in the art that an X-ray powder diffraction pattern may beobtained having one or more measurement errors depending on measurementconditions (such as equipment, sample preparation or machine used). Inparticular, it is generally known that intensities in an XRPD patternmay fluctuate depending on measurement conditions and samplepreparation. For example, persons skilled in the art of XRPD willrealise that the relative intensities of peaks may vary according to theorientation of the sample under the test and on the type and setting ofthe instrument used. The skilled person will also realise that theposition of reflections can be affected by the precise height at whichthe sample sits in the diffractometer and the zero calibration of thediffractometer. The surface planarity of the sample may also have asmall effect. Hence a person skilled in the art will appreciate that thediffraction pattern presented herein is not to be construed as absoluteand any crystalline form that provides a powder diffraction patternsubstantially identical to those disclosed herein fall within the scopeof the present disclosure (for further information, see R. Jenkins andR. L. Snyder, “Introduction to X-ray powder diffractometry”, John Wiley& Sons, 1996).

Differential Scanning Calorimetry (DSC)

Experiments were performed using a TA Instruments Q2000 DifferentialScanning Calorimeter. The DCS crucible used was a TZero aluminum panwith pinhole (diameter 0.2 mm) in the lid. A dry nitrogen purge at aconstant flow rate of 50 mL/min was maintained in the DSC cellthroughout the measurement.

EXAMPLES Example 1

Preparation of the Formulation (Small Scale)

Microcrystalline cellulose spheres were coated with one of two differentcoating suspensions of odevixibat, as indicated in Table 5 below, toobtain particles containing either 0.5% w/w or 1.5% w/w odevixibat.

TABLE 5 Amount Amount Ingredient (g/batch) (g/batch) Core:Microcrystalline cellulose spheres 700 1500 1500 (Vivapur ® MCC sphere700) Coating: Odevixibat 7.5 22.5 Hypromellose 3 mPa · s (Methocel ® E330.0 90.0 premium) Purified water^(a) 337.5 1012.5 Total (coatedparticles) 1537.5 1612.5 ^(a)Purified water is removed during thecoating and drying process.

Crystalline odevixibat was used. Typical values for the particle sizedistribution of the crystalline material were d₁₀=0.9 μm, d₅₀=4 μm andd₉₀=20 μm, wherein d₁₀, d₅₀ and d₉₀ are defined as the diameters where10%, 50% and 90%, respectively, of the particle population lies belowthese values.

Coating Suspension

The coating suspension containing odevixibat drug substance was preparedin three steps:

-   -   a) Odevixibat suspension: odevixibat drug substance was sieved        through a 0.5 mm sieve, followed by wetting in a small amount of        the water using a homogenizer (Ultra Turrax T25; 15 minutes at        approximately 6600-7000 rpm). The resulting wetted odevixibat        drug substance was then dispersed in water by means of a colloid        mill (IKA Magic Lab MKO or MK modules, 14600 rpm for 20 minutes,        gap size 1.5 rotation) until the level of agglomerates met the        in-process control acceptance limits.    -   b) Hypromellose dispersion: Hypromellose (3 mPa.$) was dispersed        in hot water with mixing, and the resultant dispersion was        cooled to room temperature.    -   c) Odevixibat coating suspension: The hypromellose dispersion        was added to the odevixibat suspension in the colloid mill and        the suspension was mixed for 4 minutes at 10000 rpm. Final        mixing was continued at low speed using a magnetic stirrer. The        odevixibat coating suspension was filtered through a 0.5 mm        sieve before use in the coating process.

The dispersion of odevixibat in the coating suspension was monitored byoptical microscopy, using a method based on European Pharmacopoeia 9.0,monograph 2.9.37, which was adjusted to be applicable for the odevixibatcoating suspension. A Leica DMLB microscope equipped with a Leica DMC2900 digital camera was used, and an objective with 10× magnification.

Samples were prepared by placing a small droplet of the coatingsuspension (using a Pasteur pipette) on a blank objective glass on topof a grid counting chamber of 4×4 test fields. A cover glass (about18×18 mm, the same size as the grid) was placed on the droplet andslightly pressed on the centre to get a thin, even sample. The diameterof the sample was comparable with the size of the cover glass.

The objective was set with magnification ×10 and the scale bar wasadjusted to 100 μm. Five replicates were scanned. The size of anyagglomerates was checked by comparing them against the scale bar in fourpredetermined test fields for each replicate. The total number ofagglomerates was calculated from 5 replicates×4 test fields, i.e. intotal 20 test fields. The coating suspension was accepted if the 20 testfields did not contain more than 5 agglomerates 50 μm, and noagglomerates 200 μm.

Coating Process

Microcrystalline cellulose (MCC) spheres were coated using theodevixibat coating suspension in a fluid bed coater with Wurster insert.The amount of coating suspension on the MCC spheres is determined byweighing. The coated particles were sieved through a 0.5 mm and 1.25 mmsieve, respectively, in order to remove fine particles as well as twins.The particles were then transferred to bulk containers and handled as adrug product intermediate.

Capsule Filling

The calculated amount of particles required for each unit dose werefilled into hard hydroxypropyl methylcellulose (HPMC) capsules (Size 0or Size 3) using an automatic capsule filler, to provide four differentstrengths: 200, 400, 600 and 1200 μg.

The 200 and 600 μg strengths are Size 0 white capsules containing 40 mgof particles having an odevixibat concentration of 0.5% w/w and 1.5%w/w, respectively. These strengths will be used for patients with aweight range of 5.0 kg to <19.5 kg in the low- (40 μg/kg) and high- (120μg/kg) dose groups of the Phase 3 clinical studies. The Size 0 capsulesare designed to be opened so that the contents can be sprinkled onto afood vehicle for administration. They are not intended to be swallowedintact.

The 400 μg and 1200 μg strengths are Size 3 white capsules containing 80mg of particles having an odevixibat concentration of 0.5% w/w and 1.5%w/w, respectively. These strengths will be used for patients with aweight range of 19.5 kg to >55.5 kg in the low- (40 μg/kg) and high-(120 μg/kg) dose groups of the Phase 3 clinical studies. The Size 3capsules are intended to be swallowed intact.

The fill weight, the amounts of odevixibat and other ingredients and thecapsule size for the different capsule strengths are shown in Table 6below.

TABLE 6 Strength 200 400 600 1200 COMPONENT μg μg μg μg odevixibatconcentration of 0.5% w/w 1.5% w/w particles Fill weight (mg)(theoretical) 40 80 40 80 Particles Microcrystalline cellulose spheres39 78 37 74 700 (Vivapur ® MCC sphere 700) Odevixibat 0.200 0.400 0.6001.200 Hypromellose 3 mPa · s 0.8 1.6 2.4 4.8 (Methocel ® E3 Premium)Capsule Hypromellose capsule, white Size 0 Size 3 Size 0 Size 3 (Vcaps ®Plus)

Example 2

Preparation of the Formulation (Larger Scale)

Microcrystalline cellulose spheres were coated with one of two differentcoating suspensions of odevixibat, as indicated in Table 7 below, toobtain particles containing either 0.5% w/w or 1.5% w/w odevixibat.

TABLE 7 Amount Amount Ingredient (kg/batch) (kg/batch) Core:Microcrystalline cellulose spheres 700 14.625 13.875 (Vivapur ® MCCsphere 700) Coating: Odevixibat 0.075 0.225 Hypromellose 3 mPa · s(Methocel ® E3 0.300 0.900 premium) Purified water^(a) 3.375 10.125Total (coated particles) 15.000 15.000 ^(a)Purified water is removedduring the coating and drying process.

Crystalline odevixibat was used. Typical values for the particle sizedistribution of the crystalline material were d₁₀=0.9 μm, d₅₀=4 μm andd₉₀=20 μm, wherein d₁₀, d₅₀ and d₉₀ are defined as the diameters where10%, 50% and 90%, respectively, of the particle population lies belowthese values.

Coating Suspension

The coating suspension containing odevixibat drug substance was preparedin three steps:

-   -   a) odevixibat suspension: odevixibat drug substance was wetted        in a small amount of the water using a homogenizer (Ultra Turrax        T25; 15 minutes at approximately 6600-7000 rpm). The resulting        wetted odevixibat drug substance was then dispersed in water by        means of a colloid mill (IKA Magic Lab MKO or MK modules, 14600        rpm for 20 minutes, gap size 1.5 rotation) until the level of        agglomerates met the in-process control acceptance limits, i.e.        d₉₀<12 μm (as determined by low-angle laser light scattering        (LALLS)).    -   b) hypromellose dispersion: Hypromellose (3 mPa.$) was dispersed        in hot water with mixing, and the resultant dispersion was        cooled to room temperature.    -   c) odevixibat coating suspension: The hypromellose dispersion        was added to the odevixibat suspension and the suspension was        mixed. Final mixing was continued at low speed using a stirrer.        The odevixibat coating suspension was filtered through a 0.5 mm        sieve before use in the coating process.

Coating Process

The obtained odevixibat coating suspension was used for coatingmicrocrystalline cellulose (MCC) spheres in accordance with the coatingprocess described in Example 1.

Capsule Filling

Capsules were prepared in accordance with Example 1. The fill weight,the amounts of odevixibat and other ingredients and the capsule size forthe different capsule strengths were as presented in Table 5 above.

Example 3

Preparation of Crystal Modification 1

Absolute alcohol (100.42 kg) and crude odevixibat (18.16 kg) werecharged to a 250-L GLR with stirring under nitrogen atmosphere. Purifiedwater (12.71 kg) was added and the reaction mass was stirred undernitrogen atmosphere at 25±5° C. for 15 minutes. Stirring was continuedat 25±5° C. for 3 to 60 minutes, until a clear solution had formed. Thesolution was filtered through a 5.0μ SS cartridge filter, followed by a0.2μ PP cartridge filter and then transferred to a clean reactor.Purified water (63.56 kg) was added slowly over a period of 2 to 3 hoursat 25±5° C., and the solution was seeded with crystal modification 1 ofodevixibat. The solution was stirred at 25±5° C. for 12 hours. Duringthis time, the solution turned turbid. The precipitated solids werefiltered through centrifuge and the material was spin dried for 30minutes. The material was thereafter vacuum dried in a Nutsche filterfor 12 hours. The material was then dried in a vacuum tray drier at25±5° C. under vacuum (550 mm Hg) for 10 hours and then at 30±5° C.under vacuum (550 mm Hg) for 16 hours. The material was isolated as anoff-white crystalline solid. The isolated crystalline material wasmilled and stored in LDPE bags.

An overhydrated sample was analyzed with XRPD and the diffractogram isshown in FIG. 2. Another sample was dried at 50° C. in vacuum andthereafter analysed with XRPD. The diffractogram of the dried sample isshown in FIG. 1.

The diffractograms for the drying of the sample are shown in FIGS. 3 and4 for 20 ranges 5-13° and 18-25°, respectively (overhydrated sample atthe bottom and dry sample at the top).

Example 4

Preparation of Crystal Modification 2 from Ethanol and Water

105.9 mg of odevixibat were weighed into a 1 mL Chromacol vessel. Amagnetic stir bar and 1.0 mL of an ethanol:water 70:30% v/v mixture wereadded and the vessel was closed with a crimped cap. The resulting slurrywas then left stirred at 25° C. for 1 week.

The wet sample was analyzed with XRPD and the diffractogram is shown inFIG. 5. Upon drying of the sample, it transformed into crystalmodification 1.

Example 5

Determination of Crystalline Fraction by Differential ScanningCalorimetry

This method quantifies the crystalline fraction of odevixibat drugsubstance in partially crystalline samples. The quantification is basedon the assumption that partially crystalline samples are binary mixturesof the crystalline hydrate and the amorphous phase of odevixibat. Thecrystalline fraction is quantified based on the melting enthalpy of ananhydrous form. This anhydrous form is the dehydrated hydrate whichspontaneously and reproducibly forms by drying the hydrate at elevatedtemperature.

5-6 mg of a sample of a crystalline or partially crystalline sample ofodevixibat was accurately weighed into a DSC crucible which was thenclosed with a perforated lid using a suitable press. The total weight ofthe DSC crucible (pan+lid+sample) was noted and the total weight of thecrucible was again determined after the DSC test. The weight loss duringthe DSC test must not be more than 5%.

The DSC test consists of three cycles:

-   -   Cycle 1: an increase in temperature from 20° C. to 120° C. at a        scanning rate of 5° C./min;    -   Cycle 2: a decrease in temperature from 120° C. to 80° C. at a        scanning rate of 10° C./min; and    -   Cycle 3: an increase in temperature from 80° C. to 200° C. at a        scanning rate of 10° C./min.

The first scan cycle dries the sample and thereby converts the hydrateform into a dehydrated hydrate (an anhydrous form). In the second scancycle, the sample is cooled down to obtain a stable baseline in thesubsequent heat-up for signal integration. The melting enthalpy isdetermined in the third scan cycle, where the sample is heated throughthe melting of the anhydrous form.

The endothermic event due to melting appears in the temperature range of140-165° C. The peak must be integrated over a sigmoidal tangentbaseline using the Sig Tangent integration function of the TA UniversalAnalysis software. The integration should start at a temperature between130° C. and 140° C., and end at a temperature between 165° C. and 175°C., depending on the actual baseline. The glass transition of theamorphous part may appear in the temperature range of 120-130° C.,depending on the actual amorphous fraction (see FIG. 6 for an example).If an irregular baseline does not allow the integration, it should beassessed whether the drying of the sample was incomplete.

The evaluation of the melting enthalpy is done by using the dry weightof the sample, which is obtained by subtracting the total weight of theDSC crucible (pan+lid+sample) after the DSC test from the total weightof the crucible before the test. The percent weight loss during the DSCscan, which is the difference between the initial weight and the dryweight divided by the initial weight, must not be more than 5%;otherwise the crystalline content of the sample cannot be calculated.The crystalline fraction expressed in weight percent is to be calculatedfrom the melting enthalpy (ΔH_(sample)) based on the following formula.The value shall be given on an integer number.

${\%{crystalline}\mspace{14mu}{content}} = \frac{{\Delta H_{{sam}\;{ple}}} + {{1.1}626}}{{0.2}815}$

Example 6

Homogeneity Monitored by LALLS

The homogeneity of odevixibat coating suspensions was studied usinglow-angle laser light scattering (LALLS). Three odevixibat coatingsuspensions intended for particles containing 0.5% w/w odevixibat wereproduced by dispersing odevixibat drug substance (16 g) in water (200 g)with an Ultra Turrax homogenizer for 7 minutes. When the drug substancewas dispersed, the homogenizer was run for an additional 8 minutes. Thehomogenizer was then rinsed with water (216 g), which was added to thesuspension.

The odevixibat suspensions were then dispersed using a wet mill (IKAMagic Lab and MK module), using the settings presented in Table 8.Methocel E3 was dispersed in hot water (85-90° C.) while mixing with anoverhead stirrer and then cooled to room temperature. The concentrationwas adjusted to 17.4% w/w by the addition of water. 368 g of theMethocel gel was charged to the odevixibat suspension and mixed usingthe wet mill for another 4 minutes at 10000 rpm. The temperature of thesuspension was checked during the process. The homogeneity of theodevixibat suspension was monitored with LALLS after 0, 5, 10, 15 and 20minutes recirculation time. The data are presented in Table 9.

TABLE 8 Suspension No. 1 2 3 Dispersion (Ultra Turrax) Time (min) 15 1515 Speed (rpm) 6800-8000 7600-8000 6600-8000 Dispersion (Wet mill) Gap(rotations) 1.5 1.5 1.0 Time (min) 20 20 20 Speed (rpm) 14600 1460014600 Cooling (MK module) Set point (° C.) N/A 25 10 Temperature (° C.)of coating 72 50 55 suspension after 20 min wet milling

TABLE 9 Recirc- Suspen- ulation LALLS (μm) sion No. time (min) d₁₀ d₅₀d₉₀ Comments 1 0 1.5 5.5 17.9 5 1.1 3.9 10.5 10 1.2 3.9 9.3 15 2.4 5.211.3 20 1.8 4.1 8.8 Sample taken after addition of Methocel 20 1.6 3.98.7 Sample taken after 5 days storage at room temperature with magneticstirring 2 0 1.5 6.5 29.1 5 1.2 4.3 11.4 10 1.1 3.8 9.3 15 1.1 3.6 8.520 1.0 3.5 8.2 Sample taken when wet milling was finished 20 1.1 3.7 8.6Sample taken after addition of Methocel 3 0 n.d. n.d. n.d. 5 0.9 3.4 8.810 0.9 3.2 7.7 15 1.1 3.7 8.5 20 1.1 3.8 8.9 Sample taken when wetmilling was finished 20 1.0 3.4 8.0 Sample taken after addition ofMethocel

Example 7

Content Uniformity

Pellets of two different strengths, 0.5% w/w and 1.5% w/w, were preparedas described in Example 1. The amount of odevixibat in a capsule wasdetermined for 30 capsules, using reversed-phase high-performance liquidchromatography (RP-HPLC). Mobile phase: 40:60 acetonitrile:acetatebuffer pH 5.5; flow rate 1.5 mL/min; column: Zorbax SB-CN (50×4.6 mm,3.5 μm). The assayed amount of odevixibat and the content uniformity arepresented in Table 10.

TABLE 10 Odevixibat concentration in particles 0.5% w/w 1.5% w/w 0.5%w/w Assay (mg/g) 4.98 14.2 4.88 Content Uniformity: RSD % (n = 30) 1.60.8 1.4

Example 8

Stability Testing at Low pH

The compatibility between particles coated with odevixibat and yoghurt,apple sauce, or fruit purée was evaluated by sprinkling about 40 mg ofcoated particles containing 0.5% w/w odevixibat (corresponding to thecontents one 200 μg capsule) onto 1 tablespoon of the food anddetermining recovery over a period of 120 minutes. The recovery ofodevixibat was determined using a reversed-phase high-performance liquidchromatography (RP-HPLC) method. Mobile phase: 40:60acetonitrile:acetate buffer pH 5.5; flow rate 1.5 mL/min; column: ZorbaxSB-CN (50×4.6 mm, 3.5 μm).

The recovery for all food samples ranged between 95% to 101% with nochange over time. Visual inspection concluded that the particles wereintact for up to 6 hours for all samples; no colour differences and nodissolved particles were observed. The results verify that patients whosprinkle the particles onto food will receive the intended dose. Asummary of the foods tested is presented in Table11, and the results ofthe recovery testing are presented in Table 12 (apple sauce), Table 13(yoghurt smoothie), and Table 14 (fruit purée).

TABLE 11 Food Brand pH Ingredients Apple sauce Eldorado 3.7 Apple 92%,sugar 7%, antioxidant (ascorbic acid), preservatives (E202) YoghurtSemper 3.9 Banana 42%, strawberry 30%, yoghurt smoothie (heat treated)20%, water, corn starch, chokeberry juice concentrate, lemon juiceconcentrate, antioxidant (ascorbic acid) Fruit purée Semper 3.9 Water,orange juice (from concentrate) 23%, apple 20%, banana 15%, rice starch,vitamin C, iron

TABLE 12 Added Nominal particle amount of Assay for Time weightodevixibat odevixibat Recovery Mean (min) (mg) (μg) (μg) (%) (%) 0 39.58201.07 199.35 99.1 99 42.35 215.14 212.28 98.7 15 40.37 205.08 187.2991.3 96 40.37 205.08 205.37 100.1 30 41.21 209.35 206.45 98.6 98 39.97203.05 197.70 97.4 60 40.50 205.74 198.14 96.3 97 40.20 204.22 199.9997.9 120 39.61 201.22 199.22 99.0 98 40.12 203.81 199.65 98.0

TABLE 13 Added Nominal particle amount of Assay for Time weightodevixibat odevixibat Recovery Mean (min) (mg) (μg) (μg) (%) (%) 0 42.07213.72 213.60 99.9 99 42.38 215.29 209.17 97.2 15 41.24 209.50 206.0198.3 97 40.42 205.33 195.58 95.2 30 40.70 206.76 199.48 96.5 96 41.96213.16 203.30 95.4 60 40.39 205.18 197.33 96.2 96 39.38 200.05 192.0696.0 120 40.99 208.23 199.05 95.6 96 39.42 200.25 192.38 96.1

TABLE 14 Added Nominal particle amount of Assay for Time weightodevixibat odevixibat Recovery Mean (min) (mg) (μg) (μg) (%) (%) 0 43.57221.34 188.05 85.0 95 37.79 191.97 200.25 104.3 15 39.74 201.88 194.8696.5 101 39.34 199.85 209.63 104.9 30 41.72 211.94 203.61 96.1 101 41.77212.19 226.27 106.6 60 42.25 214.63 208.02 96.9 96 43.39 220.42 210.7495.6 120 39.05 198.37 196.18 98.9 97 40.33 204.88 196.19 95.8

The primary degradation pathway expected for odevixibat particles, whenmixed with the specified food vehicles for administration, is acidichydrolysis of the dipeptide moiety. To evaluate the chemical stabilityof the odevixibat particles, 1 mL of phosphate buffer pH 3.0 was addedto about 200 mg of particles containing 0.5% w/w odevixibat (i.e., thecontent of five 200 μg capsules) and left at room temperature for 2hours. No degradation was observed.

Example 9

Long-Term Stability Testing

Odevixibat capsules of size 0 and size 3, prepared in accordance withExample 1, were stored in a HDPE bottle with an HDPE cap and kept at 25°C. and 60% relative humidity as the long-term storage condition, and at40° C. and 75% relative humidity as the accelerated condition. Theamounts of odevixibat, related substances and water were determinedafter 1, 3, 6, 9 and 12 months for samples stored at 25° C./60% RH, andafter 1, 3, and 6 months for samples stored at 40° C./75% RH. Theresults for capsules of strength 200 μg (Size 0) are presented in Table15; for capsules of strength 600 μg (Size 0) in Table 16; for capsulesof strength 400 μg (Size 3) in Table 17; and for capsules of strength1200 μg (Size 3) in Table 18.

TABLE 15 Stability data for capsules of strength 200 μg. AcceptanceStorage Storage period (months) Test Criteria Conditions 0 1 3 6 9 12Description White oblong 25° C./60% RH Conforms Conforms ConformsConforms Conforms Conforms hard capsule 40° C./75% RH Conforms ConformsConforms containing white to off- white pellets Assay 180-220 μg/ 25°C./60% RH 207 205 206 204 209 207 capsule 40° C./75% RH 206 206 205Related ≤4.0% 25° C./60% RH 0.53 0.51 0.48 0.47 0.55 0.57 Substances,40° C./75% RH 0.44 0.63 0.90 Total Dissolution Q = 75% at 25° C./60% RH104 101 102 102 106 106 45 mins 40° C./75% RH 99 102 101 Water Report(%) 25° C./60% RH 3.3 NT NT 3.5 NT 4.3 Content 40° C./75% RH NT NT 4.5

TABLE 16 Stability data for capsules of strength 600 μg. AcceptanceStorage Storage period (months) Test Criteria Conditions 0 1 3 6 9 12Description White oblong 25° C./60% RH Conforms Conforms ConformsConforms Conforms Conforms hard capsule 40° C./75% RH Conforms ConformsConforms containing white to off- white pellets Assay 540-660 μg/ 25°C./60% RH 576 600 596 597 603 599 capsule 40° C./75% RH 599 612 601Related ≤4.0% 25° C./60% RH 0.53 0.52 0.47 0.44 0.43 0.41 Substances,40° C./75% RH 0.45 0.48 0.73 Total Dissolution Q = 75% at 25° C./60% RH99 99 100 97 102 106 45 mins 40° C./75% RH 100 99 96 Water Report (%)25° C./60% RH 3.4 NT NT 3.3 NT 4.4 Content 40° C./75% RH NT NT 4.4

TABLE 17 Stability data for capsules of strength 400 μg. AcceptanceStorage Storage period (months) Test Criteria Conditions 0 1 3 6 9 12Description White oblong 25° C./60% RH Conforms Conforms ConformsConforms Conforms Conforms hard capsule 40° C./75% RH Conforms ConformsConforms containing white to off- white pellets Assay 360-440 μg/ 25°C./60% RH 397 395 397 400 396 399 capsule 40° C./75% RH 394 392 398Related ≤4.0% 25° C./60% RH 0.63 0.51 0.47 0.57 0.57 0.59 Substances,40° C./75% RH 0.42 0.55 0.83 Total Dissolution Q = 75% at 25° C./60% RH100 99 99 99 102 99 45 mins 40° C./75% RH 100 101 99 Water Report (%)25° C./60% RH 3.7 NT NT 3.1 NT 4.2 Content 40° C./75% RH NT NT 4.3

TABLE 18 Stability data for capsules of strength 1200 μg. AcceptanceStorage Storage period (months) Test Criteria Conditions 0 1 3 6 9 12Description White oblong 25° C./60% RH Conforms Conforms ConformsConforms Conforms Conforms hard capsule 40° C./75% RH Conforms ConformsConforms containing white to off- white pellets Assay 1080-1320 μg/ 25°C./60% RH 1191 1194 1174 1169 1196 1175 capsule 40° C./75% RH 1200 11921191 Related ≤4.0% 25° C./60% RH 0.53 0.51 0.48 0.46 0.44 0.51Substances, 40° C./75% RH 0.55 0.51 0.63 Total Dissolution Q = 75% at25° C./60% RH 99 97 99 99 98 98 45 mins 40° C./75% RH 101 100 98 WaterReport (%) 25° C./60% RH 3.5 NT NT 3.0 NT 4.1 Content 40° C./75% RH NTNT 4.3

Example 10

Blend Uniformity of Pellets

Particles of two different strengths, 0.5% w/w and 1.5% w/w, wereprepared as described in Example 2. The sieved pellets were collected ina 55 L drum, lined with a PE bag. The pellets were sampled from 10different locations of the drum with a sampling thief tip of 0.25 mL.The average sample from each location was 80 mg, corresponding to thecontent of two Size 0 capsules of 200 μg or 600 μg, or one Size 3capsule of 400 μg or 1200 μg. The content of odevixibat was determinedby RP-UPLC: Mobile phase A: 80:20 ammonium acetate buffer pH5.7/acetonitrile; Mobile phase B: 20:80 ammonium acetate buffer pH5.7/acetonitrile; flow rate 0.40 mL/min; column: Waters Acquity BEH C8100×2.1 mm, 1.7 mm; Gradient: 0 min: 60% A: 40% B, 12 min: 20% A: 80% B,13.5 min: 20% A: 80% B, 13.6 min: 60% A: 40% B, 15 min: 60% A: 40% B.The assayed amount of odevixibat and the content uniformity arepresented in Table 19.

TABLE 19 Assay for odevixibat (% of Label Claim) Sample 0.5% w/w 1.5%w/w 1 101.7 99.5 2 97.6 101.7 3 98.8 101.1 4 100.8 101.5 5 100.4 97.7 699.7 98.5 7 100.4 102.7 8 99.5 103.5 9 98.4 102.5 10 99.5 100.6 Mean99.7 100.9 Min 97.6 97.7 Max 101.7 103.5 RSD (%) 1.2 1.9

1. A pharmaceutical formulation of odevixibat, comprising a plurality ofparticles, wherein each particle is between about 0.1 and about 1.5 mmin size and contains odevixibat, or a pharmaceutically acceptable saltthereof, in an amount of from about 0.1% w/w to about 5.0% w/w based onthe total weight of the particle.
 2. The formulation according to claim1, wherein each particle contains odevixibat, or a pharmaceuticallyacceptable salt thereof, in an amount of from about 0.5% w/w to about2.0% w/w based on the total weight of the particle.
 3. The formulationaccording to claim 1, wherein each particle contains odevixibat, or apharmaceutically acceptable salt thereof, in an amount of about 0.5% w/wbased on the total weight of the particle.
 4. The formulation accordingto claim 1, wherein each particle contains odevixibat, or apharmaceutically acceptable salt thereof, in an amount of about 1.5% w/wbased on the total weight of the particle.
 5. The formulation accordingto claim 1, wherein each particle comprises a core and a coating layersurrounding the core.
 6. The formulation according to claim 5, whereinthe core does not contain odevixibat.
 7. The formulation according toclaim 5, wherein the core comprises microcrystalline cellulose.
 8. Theformulation according to claim 5, wherein the coating layer comprisesodevixibat, or a pharmaceutically acceptable salt thereof.
 9. Theformulation according to claim 5, wherein the coating layer furthercomprises a film-forming polymer.
 10. The formulation according to claim5, wherein the coating layer is sprayed onto the particles as ahomogeneous suspension of odevixibat in water.
 11. The formulationaccording to claim 10, wherein the homogenous suspension is prepared bydispersing odevixibat in water by wet milling.
 12. The formulationaccording to claim 10, wherein the homogenous suspension does notcontain agglomerates of odevixibat that are larger than 200 am.
 13. Theformulation according to claim 5, wherein the coating layer does notcontain a surfactant.
 14. The formulation according to claim 1, whereinthe particles are between about 0.1 and about 1.0 mm in size.
 15. Theformulation according to claim 1, wherein odevixibat is present as acrystalline hydrate of odevixibat.
 16. The formulation according toclaim 1, wherein odevixibat is present as crystal modification 1 ofodevixibat.
 17. The formulation according to claim 16, wherein crystalmodification 1 of odevixibat has an X-ray powder diffraction (XRPD)pattern, obtained with CuKα1-radiation, with at least specific peaks at° 2θ positions 5.6±0.2, 6.7±0.2 and/or 12.1±0.2.
 18. (canceled)
 19. Theformulation according to claim 1, which is a paediatric formulation. 20.(canceled)
 21. The method according to claim 34, wherein the liverdisease is a bile acid-dependent disease.
 22. The method according toclaim 34, wherein the liver disease is progressive familial intrahepaticcholestasis (PFIC).
 23. The method according to claim 34, wherein theliver disease is biliary atresia.
 24. A process for the preparation ofthe pharmaceutical formulation according to claim 1, comprising the stepof preparing a homogeneous aqueous suspension of odevixibat.
 25. Theprocess according to claim 24, wherein the coating suspension isprepared by dispersing odevixibat in water by wet milling.
 26. Theprocess according to claim 24, wherein the coating suspension does notcontain agglomerates of odevixibat that are larger than 200 μm.
 27. Theprocess according to claim 24, wherein the coating suspension does notcontain a surfactant.
 28. A process for the preparation of theformulation according to claim 1, comprising the steps of a) wettingodevixibat in water using a homogenizer; and b) dispersing the wettedodevixibat in water by wet milling, thereby obtaining a homogeneousaqueous suspension of odevixibat.
 29. The process according to claim 28,wherein a film forming polymer is added to the suspension obtained instep (b).
 30. (canceled)
 31. The process according to claim 24, whereinodevixibat is present as crystal modification 1 of odevixibat.
 32. Theprocess according to claim 31, wherein crystal modification 1 ofodevixibat has an X-ray powder diffraction (XRPD) pattern, obtained withCuKα1-radiation, with at least specific peaks at °2θ positions 5.6±0.2,6.7±0.2 and/or 12.1±0.2.
 33. (canceled)
 34. A method for treating orpreventing a liver disease comprising administering to a subject in needof such treatment a formulation according to claim 1.